Methods for treating viral disorders

ABSTRACT

Disclosed are methods of treating viral disorders via the administration of an inducing agent and an anti-viral agent. In one embodiment, the inducing agent and the anti-viral agent are administered for about five days, and the anti-viral agent is subsequently administered without the inducing agent for an additional period of about sixteen days for a total cycle of about 21 days.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S. patentapplication Ser. No. 13/915,092 filed Jun. 11, 2013, which is acontinuation application of U.S. patent application Ser. No. 12/890,042filed on Sep. 24, 2010, which claims the benefit under 35 U.S.C. §119(e)of U.S. Provisional Application No. 61/245,529, filed Sep. 24, 2009, andU.S. Provisional Application No. 61/295,663, filed Jan. 15, 2010, thecontents of each of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 6, 2013, isnamed 701586-069032_SL.txt and is 1,244 bytes in size.

BACKGROUND OF THE INVENTION

A growing number of cellular disorders such as neoplastic malignancieshave been found to contain viral genetic sequences or virus particles inthe anomalous cells. For a large number of these disorders, the presenceof the virus is believed to be a causative or at least contributoryinstrument. Representative members of many of the known families ofviruses have been found in such cells including members of the herpesfamily of viruses, the polyomaviruses, and the hepatitis viruses.Epstein-Barr virus (EBV), a 172 kb herpes virus, is often foundintimately associated with both mature and immature B cells. EBV is acommon and worldwide pathogen. Childhood infection is asymptomatic.About 50% of individuals with delayed exposure develop a self-limited,lymphoproliferative syndrome referred to as infectious mononucleosis.EBV is also detected in 2 endemic tumors, African Burkitt's lymphoma(BL) (Henle, G., et al. 1985 Proc. Natl. Acad. Sci. USA 58:94-101) andnasopharyngeal carcinoma (NPC) (Henle, W., et al. 1985 Adv. Viral Onco.5:201-38), as well as gastric carcinoma, breast cancers and sarcomas.Recently, some T-cell and B-cell lymphomas, as well as about 50% ofHodgkin's lymphomas, have been found to contain EBV (Weiss, L. M., etal. 1989 N. Engl. JMed. 320:502).

EBV undergoes lytic replication after initial infection of oropharyngealepithelia. The linear form genome is duplicated, packaged into the viralcapsid, and extruded from the cell by budding or lysis. One hundredviral proteins are synthesized during this lytic stage of the virus lifecycle. In contrast, normal B cells incubated with EBV in vitro areefficiently immortalized and develop into continuously growinglymphoblastoid cell lines (LCLs). The cellular events that regulatethese distinct outcomes are as yet unclear. In immortalized cells, thegenome circularizes, amplifies, and replicates coordinate with, anddependent upon, cell division. Because no viral particles are produced,infection is considered to be latent, and EBV persists in the cells forlife. Outgrowth of latently infected B cells is prevented by T cellimmune surveillance. In immortalizing latent infection, only 11 geneproducts are detected, including 6 nuclear antigens (EBNA-1, -2, -LP,-3A, -3B, -3Q), 3 membrane proteins (LMP-1, LMP-2A, LMP-2B) and twosmall, non-poly(A) RNAs (EBER-1 and EBER-2) (Miller, G., et al. 1990Epstein-Barr Virus: Biology, Pathogenesis and Medical Aspects, RavenPress, N.Y.). In EBV(+) tumors such as Burkitt's lymphoma, neoplasticgenetic events have often superseded the requirement for viralimmortalizing functions, and gene expression may be limited to EBNA-1(Rowe, M., et al. 1987 EMBO J. 6:2743-51). Virus tropism is deteiminedby complement receptor type 2 which mediates attachment of the envelopeprotein gp350/2204 to B and some T lymphocytes, follicular dendriticcells and epithelial cells.

African Burkitt's lymphoma is characterized by rapid growth of the tumorat non-lymphoid sites such as the jaw or the retroperitoneum. The tumoris of B cell origin and is closely related to the small non-cleavedcells of normal lymphoid follicles. Biopsy specimens from AfricanBurkitt's lymphoma invariably contain the EBV genome and are positivefor EBNA (Magrath, I. 1986 Epstein-Barr Virus and Associated Diseases,pp. 631-43, M. Ninjhoff Publishing, Boston). This contrasts with thenon-African Burkitt's lymphoma, in which only 15% to 20% of the tumorscontain the EBV genome. EBV has a worldwide distribution and infectsmost (more than 90%) individuals before adulthood. The clustering ofBurkitt's lymphoma in the equatorial belt of East Africa remainsunexplained. It has been hypothesized that alterations of the immunesystem, possibly due to hyperstimulation by endemic malaria, may play animportant role in the outcome of an EBV infection to individuals in thisregion (Moss, D. J., et al. 1983 Int. J. Cancer 31: 727-32). Individualsfrom this region show impairment in virus-specific cytotoxic T-cellactivity. Normally, it is the T-cell response to EBV infection thatlimits B-cell proliferation, and this T-cell response is directlystimulated by EBV (zur Hausen, H., et al. 1970 Nature 228: 1056-58). Ithas been postulated that the failure of the T-cell immune response tocontrol this proliferation could lead to excessive B-cell proliferationand, as such, provide a suitable background for further mutation,oncogenic transformation, and lymphomagenesis.

A scenario has been suggested for the involvement of EBV in the etiologyof African Burkitt's lymphoma (Klein, G. 1979 Proc. Natl. Acad. Sci. USA76:2442-46). The first step involves the EBV-induced immortalization ofB lymphocytes in a primary infection. The second step involves thestimulated proliferation of EB V(+) B cells. This step is facilitated inthe geographic areas where Burkitt's lymphoma is endemic (presumablybecause of the presence of malaria), through B-cell triggering and thesuppression of T-cells involved in the control of the proliferation ofEBV-infected cells. This pool of cells becomes increased in size as atarget cell population for random chromosomal rearrangements. The thirdand final step is the reciprocal translocation involving a chromosomallocus with an immunoglobulin gene and the c-myc gene on chromosome 8.This leads to the deregulation of the c-myc gene, to the development ofthe malignant clone, and to the appearance of a tumor mass (Klein, G.,et al. 1985 Nature 315: 190). Alternative scenarios have been proposed,in which the order of the steps is rearranged such that the B-cellactivation by malaria precedes the chromosomal translocation and isfollowed by EBV infection. Regardless, the components of these twoscenarios each account for the geographic distribution of Burkitt'slymphoma, the critical involvement of EBV in lymphomagenesis, and theeventual selection and clonal outgrowth of a population of cells withthe critical translocation involving the deregulation of the c-myc geneon chromosome 8.

For more than 20 years, a role for EBV in the pathogenesis of Hodgkin'sdisease (HD) was postulated based on epidemiologic evidence linkingHodgkin's patients with EBV seropositivity and elevated EBV titers(Evans, A. S., et al. 1984 Int. J. Cancer 34: 149). A number of studieshave found an increase (2-5 fold) in the incidence of HD afterinfectious mononucleosis. However, some Hodgkin's patients wereseronegative for EBV, and the association between EBV and Hodgkin'sdisease remained speculative until 1987. I n that year, moleculargenetic analysis demonstrated that some Hodgkin's tissues containedmonoclonal EBV DNA and that the virus was localized to Reed-Sternberg(RS) cells (the malignant cells in HD). Subsequent immunohistochemicaland serologic data support an association between EBV and Hodgkin'sdisease and confirm the localization of the virus to cytologicallymalignant-appearing RS cells and variants (Jiwa, N. M., et al. 1992Histopathology 21:51). EBV also infects variable numbers of small B andT lymphocytes in the reactive inflammatory cell infiltrate that composesthe bulk of Hodgkin's tissues (Weiss, L. M., et al. 1991 Am. J. Path.139: 1259). Clonal and non-clonal EBV genomes are present in Hodgkin'sdisease. Expression of the oncogene LMP (latent membrane protein) isseen in RS cells. In HD, the region of the (viral) BNLF1 oncogene codingfor the amino terminal and transmembrane domains (associated withoncogenic function) of LMP appears to be homogeneous, whereas the regioncoding for the intracytoplasmic (carboxyl terminal) domain of LMP isheterogeneous. Cytological similarities between RS cells andimmunoblasts of known EBV-induced infectious mononucleosis andEBV-induced AIDS-related lymphomas are consistent with the hypothesisthat the EBV-BNLFI oncogene is an inducer of morphological features ofRS cells. Whether chromosomal integration of EBV DNA is an importantfactor in activation of such a transforming activity remains to beelucidated. Therefore, the RS cells appear to be derived fromlymphocytes beyond the pre-B-cell or common thymocyte stage, which mayor may not subsequently become infected by EBV.

The high prevalence of EBV in Hodgkin's disease implies an etiologicrole for the virus in some cases of Hodgkin's tumorigenesis. Thispathogenetic theory is supported by the monoclonality of EBV DNA inthese tumors (Gulley, M. L., et al. 1994 Blood 83: 1595-602). In oneseries, monoclonal EBV DNA was detected in all 17 cases havingEBNA1-positive RS cells. Because tumor-associated viral DNA ismonoclonal, it is likely that virus infection preceded clonal expansion.This reinforces the hypothesis that the virus is not an innocentbystander, but, rather, plays a role in the pathogenesis of theHodgkin's disease and the other tumor types in which it is found (Neri,A., et al. 1991 Blood 77: 1092). The observation of EBNA1 expression inthe RS cells of clonally-infected cases indicates that the clonal virusis localized to these cells and suggests that Hodgkin's disease resultsfrom the transformation of an EBV-competent cell. Other studies suggestthat this virus is a modulating rather than an etiologic agent in aconsiderable proportion of HD cases.

Investigations into the biology of EBV infection have shown that onlyone viral particle successfully infects a given cell. Once the viral DNAis established inside the cell, it circularizes and reproduces itself toyield multiple identical copies of viral DNA (Hurley, E. A., et al. 1988J. Exp. Med. 168:2059). In this way, tumors derived from infected cellscan have multiple copies of EBV per cell, while maintaining clonal viralDNA structure. The average amount of clonal EBV DNA in Hodgkin's diseasetissues varied from 0.5 to 5 copies per cell. Because RS cells comprisedonly a small fraction (<1%) of all HD tissue cells, the content of EBVDNA in each RS cell is estimated to be at least 100 times higher thanthe measured average copy number per cell, or at least 50 copies ofviral DNA per RS cell. This is comparable with, or greater than, theviral burden in infected non-Hodgkin's lymphomas. The high copy numberof EBV in RS cells may relate to the pathobiology of this complexlymphomatous disorder. In agreement with these studies, EBV DNA isabundant and monoclonal in infected RS cells. The presence of EBV in RScells was strongly and independently linked to mixed cellularityhistology and Hispanic ethnicity.

Clonally-integrated EBV is found in association with T-cell lymphomas,as well as B-cell lymphomas. Currently, three populations oftissue-restricted T lymphocytes have been recognized: mucosa-associated,cutaneous, and nodal T lymphocytes. T-cell lymphomas arising fromdifferent sites, but with similar morphology, may show differences inlymphomagenesis and in expression of oncogenes, adhesion molecules,presence of certain DNA/RNA viral sequences, and in clinicalpresentation and behavior. Primary cutaneous CD30(+) large cell, T-celllymphomas often remain localized to the skin for a long time, express aunique cutaneous lymphocyte antigen (CLA), known as the skin-homingreceptor, have been postulated to be associated with the presence ofhuman T-cell leukemia/lymphoma virus type I (HTLV 1), and have a goodclinical course (Beljaards, R. C., et al. 1993 Cancer 71:2097). Incontrast, morphologically similar T-cell lymphomas of nodal origin oftenbehave more aggressively, are CLA-negative, and have been associatedwith the presence of EBV (de Bruin, P. C., et al. 1993 Histopathology23: 127). There was no relation between primary cutaneous T-celllymphoma and EBV.

Infection of T cells by EBV most likely occurs via CR2 or CR2-likereceptors (Tsoukas, C. D., et al. 1993 Immunol. Today 14:56). The closecontact between T cells and the upper respiratory tract epithelium,known for its reservoir function for EBV, probably make T cells in thisregion more vulnerable for EBV infection. The finding that EBV can befound in almost all tumor cells in most cases of primary extra-nodal,and especially nasal, T-cell lymphoma, in contrast to primary nodalT-cell lymphoma, where the number of EB V-infected neoplastic cellsvaries greatly between the cases, argues for an etiologic role for EBVin these cases. Moreover, these cases often express LMP-1, known for itstransforming and oncogenic properties in vitro and are reported to bemonoclonal for EBV (Su, I., et al. 1991 Blood 77:799). Thus, there aresite-restricted etiologic differences between morphologically identicalT-cell lymphomas, of which EBV might be one of many factors.

EBV-induced lymphoproliferative disease or lymphoma has animmunodeficiency incidence in U.S. of about 10,000 B-cell, EBV(+)lymphoma patients per year. EBV is very commonly associated withlymphomas in patients with acquired or congenital immunodeficiencies.These lymphomas can be distinguished from the classical Burkitt'slymphomas in that the tumors may be polyclonal. Tumors also do notdemonstrate the characteristic chromosomal abnormalities of Burkitt'slymphoma described earlier. The pathogenesis of these lymphomas involvesa deficiency in the effector mechanisms needed to control EBV−transformed cells. The prototypic model for this disease has been theX-linked lymphoproliferative (XLP) syndrome (Purtilo, D. T., et al. 1982Am. J. Med. 73:49-56). Patients with XLP who develop acute infectiousmononucleosis exhibit the usual atypical lymphocytosis and polyclonalelevation of serum immunoglobulins and increases in specific antibody toVCA and to EA. During these infections, patients with XLP fail to mountand sustain an anti-EBNA response after acute EBV infection. The uniquevulnerability of males with XLP to EBV infection is most likely due toan inherited immune regulatory defect that results in the failure togovern the cytotoxic T cells and NK cells required to cope with EBV.

The herpes virus family members are capable of bypassing thebutyrate-mediated block, which is probably due to the role of viralearly genes in DNA synthesis, such as the viral DNA polymerase,DNA-binding protein, and helicase genes (Shadan, F. F., et al. 1994 J.Virol. 68:4785-96). Butyrate treatment has been reported to result inthe induction of the major CMV major immediate-early protein (IEP) byactivating the IEI promoter via cellular factors in a human epithelialthyroid papilloma carcinoma cell line and in cultured endothelial cellsunder conditions that are conducive to terminal differentiation(Villarreal, L. P. 1991 Microbiol. Rev. 55:512-42). Similarly, EBV earlyantigen is induced by butyrate in the P3HR-1 cell line, as well as inRaji and NC37 cell lines. These results indicate that butyrate exertssome of its effects on viral growth at the level of gene transcription.This conclusion is also supported by the observation that butyrateactivates the long terminal repeat-directed expression of humanimmunodeficiency virus and induces the Moloney murine sarcoma virus viaa putative butyrate response enhancer-promoter element (Bohan, C., etal. 1987 Biochem. Biophys. Res. Commun. 148:899-907; Tang, D. C., et al.1992 Biochem. Biophys. Res. Commun. 189: 141-47; Yeivin, A., et al. 1992Gene 116: 159-64). Therefore, butyrate appears to be associated with ageneral induction of early viral proteins. Butyrate has been reported toexert additional cytostatic effects such as G2/M blockage and anti-viralactivity against RNA viruses.

Unlike other members of the herpes virus family, EBV is resistant to theantiviral agent ganciclovir, because of low levels of viral thymidinekinase. Acyclovir and ganciclovir have also been used to treat AIDSpatients, many of whom had an active EBV infection. During treatment,regression of hairy leukoplakia, an EBV disorder, was inadvertentlyobserved while latent EBV infection was unaffected. Additional studiesdemonstrated that even when virus production is minimal, expression ofmany EBV genes active during the lytic cycle, such as thymidine kinase,can be induced. Therefore, exposure of EBV-transformed B-cells or tumorcells to arginine butyrate induces EBV-TK and renders them sensitive toganciclovir.

Like herpes simplex virus (HSV) and varicella-zoster virus (VZV), EBVencodes a thymidine kinase enzyme localized to the BamHI, X fragment ofthe genome. In a rate-limiting step, the TK converts nucleoside analogsto their monophosphate form. Cellular enzymes complete their conversionto biologically-active triphosphates. A viral DNA polymerasepreferentially incorporates the toxic metabolites into viral DNA,leading to premature termination of the nascent DNA. ACV is a purinenucleoside analog with a linear side chain replacing the cyclic sugar ofguanosine. GCV differs from ACV in the addition of a hydroxymethyl groupto the side chain. However, ACV and GCV differ in functional assays.Whereas HSV TK preferentially phosphorylates ACV, EBV-TK preferentiallyphosphorylates GCV. Furthermore, because GCV triphosphate accumulates tohigher levels and persists for longer periods in infected cells thanACV, GCV produces more interference with cellular DNA synthesis thanoccurs with ACV. In one study, selective toxicity of GCV for cellsexpressing HSV-TK was utilized to promote tumor killing in the CNS.Rapidly dividing murine glioma cells were infected in vivo with anamphotropic retrovirus containing HSV-TK. Animals were treated with GCV,which killed TK+ tumor cells, sparing adjacent normal cells thatreplicated too slowly for efficient infection and viral TK expression.

EBV-induced lymphomas are associated with immunosuppression. Patientswith iatrogenic immunodeficiencies, such as organ transplant recipients,are also at an increased risk for lymphomas, and these lymphomas oftencontain EBV DNA and EBNA. Also, patients with AIDS are at a higher riskfor developing polyclonal lymphomas associated with EBV. EBV-associatedlymphoproliferative disease (EBV-LPD) is characterized by activelyproliferating EBV(+) B-cells, frequently without overt malignant change.These immunoblastic lymphoma-like lesions have been identified in avariety of transplant patients, in patients with congenitalimmunodeficiency, and in patients infected with HIV (Cohen, J. I. 1991Medicine 70: 137-60). These so called post-transplantlymphoproliferative disorders (PTLD) are observed after all transplants,including kidney, bone marrow, heart, liver, and lung transplantation.This increased incidence of EBV-LPD in this setting is likely due to theaggressive immunosuppression required after these transplants.

Although there are case reports of administration of inducing agentssuch as butyrates to patients for the treatment of malignancies(Novogrodsky, A., et al. 1983 Cancer 51:9-14; Miller, A. A., et al. 1987Eur. J. Cancer Clin. Oncol. 23: 1283-89), as well as of administrationof anti-viral agents for the treatment of viral disorders, there are notreatments requiring the administration of both agents and involving ashort course regimen.

Thus, there remains a need for effective regimens for treatment ofviral-associated disorders.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a method for treating aviral disorder in a subject, comprising providing said subject with atleast one cycle of therapy, said cycle of therapy comprising: i)administering to the subject over a first period an inducing agent toinduce expression of a viral gene product in a virus-infected cell ofthe subject and an anti-viral agent whose anti-viral activity isdirected to the viral gene product expressed, wherein said first periodof time is less than or equal to one-half of the length of the cycle,and further wherein said inducing agent and said anti-viral agent areadministered in the same or separate compositions; and ii) administeringsaid anti-viral agent to the subject for a second period, wherein saidsecond period is the remainder of the cycle. The first wherein periodmay be less than or equal to about 5 days, and the second period may beat least about 5 days and less than or equal to about 16 days. Invarious aspects, the method is a method for killing virus-infected cellsin vivo.

In another embodiment of a method of the invention, the subject isprovided less than or equal to about six cycles of therapy. In yetanother embodiment of a method of the invention, the subject is providedmore than or equal to six cycles of therapy. In still another embodimentof a method of the invention, the inducing agent is administered to thesubject via continuous infusion over the first period of time, which, inanother embodiment, is less than or equal to about five days. In stillanother embodiment of a method of the invention, the inducing agent isadministered to the subject via once daily infusion over the firstperiod of time, which, in another embodiment, is less than or equal toabout five days. In still another embodiment of a method of theinvention, the inducing agent is administered to the subject at leastonce over the first period of time, which, in another embodiment, isless than or equal to about five days.

In another embodiment of a method of the invention, the anti-viral agentis administered intravenously to the subject about one to about twotimes per day over the first period of time of the cycle and orallyabout one to about two times per day over the remainder of the cycle. Instill another embodiment of a method of the invention, the inducingagent is administered at a dose of about 0.1 to about 2000 mg/kg/day orabout 1 to about 100 mg/m²/day. In still another embodiment of a methodof the invention the inducing agent is administered at a dose of about1000 mg/kg/day. In still another embodiment of a method of theinvention, the inducing agent is administered at a dose of about 5mg/m²/wk.

In another embodiment of a method of the invention, the inducing agentis selected from the group consisting of a short-chain fatty acid, ashort-chain fatty acid derivative, a histone deacetylase (HDAC)inhibitor, a DNA methyltransferase inhibitor, a proteasome inhibitor, aphorbol ester, an oxidized phorbol ester, ceramide, bryostatin, acytokine, and combinations thereof. In another aspect, the inducingagent is selected from arginine butyrate, PMA (phorbol myristateacetate), TSA (trichostatin A), 2,2-dimethyl butyrate, panobinostat(LHB589), apicidin, MS-275, and largazole. In yet another embodiment ofa method of the invention, the inducing agent is arginine butyrate.

In another embodiment of a method of the invention, the anti-viral agentis selected from the group consisting of an interferon, an amino acidanalog, a nucleoside analog, an integrase inhibitor, a proteaseinhibitor, a polymerase inhibitor, and a transcriptase inhibitor. Instill another embodiment of a method of the invention, the anti-viralagent is acyclovir (ACV), ganciclovir (GCV), famcyclovir, penciclovir,foscarnet, ribavirin, zalcitabine (ddC), zidovudine (AZT), stavudine(D4T), larnivudine (3TC), didanosine (ddl), cytarabine,dideoxyadenosine, edoxudine, floxuridine, idozuridine, inosine pranobex,2′-deoxy-5-(methylamino)uridine, trifluridine or vidarabine. In stillanother embodiment of a method of the invention, the anti-viral agent isganciclovir.

In another embodiment of a method of the invention, the inducing agentand the anti-viral agent are administered separately.

In another aspect, the invention provides a method for killingvirus-infected cells in vivo, comprising providing a subject with atleast one cycle of therapy, said cycle of therapy comprising: i)administering to the subject over a first period of time apharmaceutical composition comprising an inducing agent to induceexpression of a viral gene product in a virus-infected cell of thesubject and an anti-viral agent whose anti-viral activity is directed tothe viral gene product expressed, wherein said first period of time isless than or equal to one-half of the length of the cycle; and ii)continuing the administration of the anti-viral agent to the subject forthe remainder of the cycle, thus killing the virus-infected cells invivo. The administration periods may be about 5 days and about 16 days,for a cycle of about 21 days, in various embodiments.

In another embodiment of a method of the invention, the virus-infectedcells are lymphocytes. In still another embodiment of a method of theinvention, the virus-infected cells contain an integrated or episomallatent virus. In still another embodiment of a method of the invention,the virus-infected cells contain a herpes virus, a human T cell or Bcell leukemia virus, an adenovirus, or a hepatitis virus. In stillanother embodiment of a method of the invention, the herpes virus is anEpstein-Barr virus (EBV) or a Kaposi's-associated human herpes virus. Instill another embodiment of a method of the invention, the leukemiavirus is a human immunodeficiency virus (HIV) or a human T-cellleukemia/lymphoma virus (HTLV).

In various embodiments, the viral disorder is a neoplasia associatedwith viral infection. Alternatively, the neoplasia is selected from thegroup consisting of lymphoma, Hodgkin disease, Burkitts lymphoma,post-transplantation lymphoproliferative disease, viral associatedlymphoproliferative disease, hemophagocytic syndrome, nasopharyngealcarcinoma, gastric carcinoma, or breast cancer.

In various embodiments, the viral disorder is a non-malignant viraldisorder.

In another embodiment of a method of the invention, the viral geneproduct regulates viral gene expression. In yet another embodiment of amethod of the invention, the viral gene product is a viral enzyme, anoncogene or proto-oncogene, a transcription factor, a protease, apolymerase, a reverse transcriptase, a cell surface receptor, astructural protein, a major histocompatibility antigen, a growth factor,or a combination thereof. In yet another embodiment of a method of theinvention, the viral enzyme is a thymidine or protein kinase. In yetanother embodiment of a method of the invention, the gene productexpressed sensitizes the infected-cells to the anti-viral agent. Invarious embodiments, induction of gene products that target the cell fordestruction by the immune system are excluded such that the therapeuticaction is essentially through the activity of the anti-viral agent.

In another aspect, the invention provides a method for treating acellular disorder resulting from and/or associated with viral infectionin a subject, comprising providing said subject with at least one cycleof therapy, said cycle of therapy comprising: i) administering to thesubject over a first period of time a pharmaceutical compositioncomprising an activator and an anti-viral agent, wherein the activatoris administered in an amount sufficient to activate expression of alatent virus episomal or integrated into proliferating cells of thesubject, wherein said first period of time is less than or equal toone-half of the length of the cycle; and ii) continuing theadministration of the anti-viral agent to the subject for the remainderof the cycle, thus treating the cellular disorder resulting from and/orassociated with viral infection in the subject.

In another aspect, the invention provides a method for treating anHIV-associated disorder in a subject, comprising providing said subjectwith at least one cycle of therapy, said cycle of therapy comprising: i)administering to the subject over a first period of time: a) ananti-viral agent, and b) an inducing agent in an amount sufficient toinduce expression of a viral gene product selected from the groupconsisting of EBV-thymidine kinase and HIV reverse transcriptase; andii) continuing the administration of the anti-viral agent to the subjectfor the remainder of the cycle.

Other objects and advantages of the invention are set forth in part inthe description of the invention that follows and, in part, will beapparent from this description or may be learned from the practice ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate CT scans of tumor reduction after treatment witharginine butyrate (AB) and GCV. FIGS. 1A and 1B show pre- andpost-treatment, respectively.

FIGS. 2A-2B illustrate results from toxicity assays withanti-herpesvirus drugs ganciclovir (GCV) and penciclovir (PCV). FIGS. 2Aand 2B show results from various concentrations of GCV and PCV. FIGS. 2Cand 2D show results from GCV and PCV used in combination with NaB.

FIGS. 3A-3B illustrate the results from analysis of efficacy ofanti-virals using short chain fatty acids as inducing agents. FIG. 3Ashows cell count results after using NaB. FIG. 3B shows results from VA.FIG. 3C shows fold of TK expression induced.

FIGS. 4A-4B illustrate results from analysis of efficacy of anti-viralsusing hydroxamic acids as inducing agents. FIG. 4A shows cell countresults after using scriptaid. FIG. 4B shows fold of TK expressioninduced.

FIGS. 5A-5B illustrate results from analysis of efficacy of anti-viralsusing SAHA (Vorinostat) as an inducing agent. FIG. 5A shows cell countresults after using SAHA. FIG. 5B shows fold of TK expression induced.

FIGS. 6A-6B illustrate results from analysis of efficacy of anti-viralsusing LHB589 (Panobinostat) as an inducing agent. FIG. 6A shows cellcount results after using LHB589. FIG. 6B shows fold of TK expressioninduced.

FIG. 7 illustrates results from analysis of efficacy of anti-viralsusing PXD 101 which induced a high level of TK expression at the 5 μMconcentration.

FIG. 8 illustrates results from analysis of efficacy of anti-viralsusing oxamflatin as an inducing agent. FIG. 8 shows cell count resultsafter using oxamflatin.

FIG. 9 illustrates results from analysis of efficacy of anti-viralsusing a cyclic tetrapeptide as an inducing agent. FIG. 9 shows cellcount results after using apicidin.

FIGS. 10A-10C illustrate results from analysis of efficacy ofanti-virals using a benzamide (MS-275) as an inducing agent. FIG. 10Ashows cell count results after using MS-275. FIG. 10B shows fold of TKexpression induced. FIG. 10C shows cell count results after treatingcells in combination for shorter time periods.

FIGS. 11A-11B illustrate chemical structures of largazole compoundsused. Shown in both FIGS. 11A and 11B.

FIGS. 12A-12F illustrate results from analysis of efficacy ofanti-virals using largazoles as an inducing agent. FIGS. 12A, 12B, 12C,12D and 12E show cell count results after using various largazolecompounds. FIG. 12F shows fold of TK expression induced from variouslargazole compounds.

FIG. 13 illustrates results from treatment of an HIV-1-infected monocytecell line with combination therapy. Viral release (p24 release) wasmeasured through optical density (OD) measurement.

FIG. 14 illustrates results from treatment of an HIV-1-infected monocytecell line with combination therapy. Viral release (p24 release) wasmeasured through optical density (OD) measurement and then convertedinto pg of protein.

DETAILED DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to methods for the treatment and prevention of viral diseasesand disorders. It has now been found that the relevant effect of aninducing agent lasts long after it has been administered and, even afterthe inducing agent is no longer detectable in the subject's plasma. Inthe currently described treatment regimen, one contemplated dose is 1000mg/kg/day for 5 days (i.e., 5000 mg/kg) of inducing agent. It issurprising, for example, in virus-associated neoplastic disorders, thatthe dose of drug and the time span of its administration can be cut downdramatically—both the total amount of inducing agent administered, andthe time span over which it is given.

In one aspect, the invention provides methods for treatingvirus-associated disorders. The methods comprise providing a subjectwith at least one cycle of therapy comprising administering over a firstperiod of time a therapeutic agent, such as an anti-viral agent, and aninducing agent that stimulates the expression of a specific gene, suchas a virus-associated gene, that renders the infected cell susceptibleto further therapeutic treatment via a therapeutic agent such as ananti-viral agent, and subsequently, continuing to administer thetherapeutic agent for the remainder of the cycle. The inducing agent isadministered in a short course regimen (i.e., over the first period oftime less than or equal to one half of the length of the cycle oftherapy over which the therapeutic agent is given).

The inducing agents may induce expression of certain viral geneproducts. Gene products that can be induced by agents of the inventioninclude, but are not limited to, viral proteins such as viral thymidinekinase and the BGLF4 protein kinase of EBV-infected cells. Expression ofthese products can be used in the treatment and prevention of viraldisorders.

The therapeutic agents may comprise anti-viral agents that, for example,in combination with certain inducing agents, destroy virus-infectedcells. Effective anti-viral agents that can be used include substrateanalogs such as nucleoside analogs, polymerase inhibitors, andtranscriptase inhibitors. Cells that demonstrate induced expression orproliferation are targeted and destroyed.

Another embodiment of the invention is directed to a method fordestroying, killing or otherwise severely crippling virus-infected cellsby treating said cells with an inducing agent, to induce the activity ofa gene product, and an anti-viral agent whose anti-viral activity isdirected to the activity of the gene product induced, over a firstperiod of time less than or equal to one half of the duration of a cycleof therapy, followed by treatment with the anti-viral agent alone overthe remainder of the cycle of therapy. The combination portion of thetreatment course is, thus, a short course regimen.

Preferably, the gene product is a viral enzyme that relates to a basicand necessary process of the virus such as virus adsorption, cellpenetration, fusion, uncoating, reverse transcription, integration, DNAreplication, viral interference, viral transcription, the switch fromearly to late expression, the latent or lytic phases, the switch from alatent to lytic phase, defective-interfering particle production, virusassembly, capsid packaging, the generation of virus-specific membrane,virus budding and virus secretion. The activity of one or more of theseprocesses may be enhanced by one or more of the inducing agents, and theenhanced activity is then targeted by one or more anti-viral agents. Thecombination treatment as part of the cycle of therapy is more effectivethan conventional treatment with anti-viral agents alone or simplyallows for the administration of therapeutically effective amounts ofthe anti-viral agent that are less than what would be consideredeffective with conventional treatment regimens.

Another embodiment of the invention is directed to a method for treatinga viral disorder in a patient comprised of administering an activatorand an anti-viral agent to the patient, wherein the activator isadministered in an amount sufficient to activate expression of a latentvirus integrated into proliferating cells of the patient. The treatmentregimen is as described above for the methods of the invention. Usefulactivators include phorbol ester, an oxidized phorbol ester, ceramide,bryostatin, an inducing agent, or a combination thereof. The activatorshould be administered in an amount sufficient to activate, for example,protein kinase C, an oncogene, a thymidine kinase or other viralkinases, AP-1, AP-2, Sp-1 or NF-KB.

A. DEFINITIONS

The terms “viral” and “virus-associated” with reference to disorders areused interchangeably throughout the instant specification.

The term “treating”, as used herein, refers to altering the diseasecourse of the subject being treated. Therapeutic effects of treatmentinclude, without limitation, preventing occurrence or recurrence ofdisease, alleviation of symptom(s), diminishment of direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

The term “pharmaceutically acceptable excipient”, as used herein, refersto carriers and vehicles that are compatible with the active ingredient(for example, a compound of the invention) of a pharmaceuticalcomposition of the invention (and preferably capable of stabilizing it)and not deleterious to the subject to be treated. For example,solubilizing agents that form specific, more soluble complexes with thecompounds of the invention can be utilized as pharmaceutical excipientsfor delivery of the compounds. Suitable carriers and vehicles are knownto those of extraordinary skill in the art. The term “excipient” as usedherein will encompass all such carriers, adjuvants, diluents, solvents,or other inactive additives. Suitable pharmaceutically acceptableexcipients include, but are not limited to, water, salt solutions,alcohol, vegetable oils, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid monoglycerides and diglycerides, petroethralfatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc.The pharmaceutical compositions of the invention can also be sterilizedand, if desired, mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like, which do not deleteriously react withthe active compounds of the invention.

The term “subject” as used herein refers to a vertebrate, preferably amammal, more preferably a primate, still more preferably a human.Mammals include, without limitation, humans, primates, wild animals,feral animals, farm animals, sports animals, and pets.

The term “obtaining” as in “obtaining the composition” is intended toinclude purchasing, synthesizing, or otherwise acquiring the composition(or agent(s) of the composition).

The terms “comprises”, “comprising”, are intended to have the broadmeaning ascribed to them and can mean “includes”, “including” and thelike.

The invention can be understood more fully by reference to the followingdetailed description and illustrative examples, which are intended toexemplify non-limiting embodiments of the invention.

B. VIRUSES AND VIRAL (INCLUDING VIRUS-ASSOCIATED) DISORDERS

The methods and compositions of the provided invention can be used totreat and/or prevent viral infections. The virus causing the infectioncan be a member of the herpes virus family, a human immunodeficiencyvirus, parvovirus, or coxsackie virus. A member of the herpes virusfamily can be herpes simplex virus, herpes genitalis virus, varicellazoster virus, Epstein-Barr virus, human herpesvirus 6, orcytomegalovirus.

The methods and compositions described herein can be used to treatand/or prevent infections caused by any virus, including, for example,Abelson leukemia virus, Abelson murine leukemia virus, Abelson's virus,Acute laryngotracheobronchitis virus, Adelaide River virus, Adenoassociated virus group, Adenovirus, African horse sickness virus,African swine fever virus, AIDS virus, Aleutian mink disease parvovirus,Alpharetrovirus, Alphavirus, ALV related virus, Amapari virus,Aphthovirus, Aquareovirus, Arbovirus, Arbovirus C, arbovirus group A,arbovirus group B, Arenavirus group, Argentine hemorrhagic fever virus,Argentine hemorrhagic fever virus, Arterivirus, Astrovirus, Atelineherpesvirus group, Aujezky's disease virus, Aura virus, Ausduk diseasevirus, Australian bat lyssavirus, Aviadenovirus, avian erythroblastosisvirus, avian infectious bronchitis virus, avian leukemia virus, avianleukosis virus, avian lymphomatosis virus, avian myeloblastosis virus,avian paramyxovirus, avian pneumoencephalitis virus, avianreticuloendotheliosis virus, avian sarcoma virus, avian type Cretrovirus group, Avihepadnavirus, Avipoxvirus, B virus, B 19 virus,Babanki virus, baboon herpesvirus, baculovirus, Barmah Forest virus,Bebaru virus, Berrimah virus, Betaretrovirus, Birnavirus, Bittner virus,BK virus, Black Creek Canal virus, bluetongue virus, Bolivianhemorrhagic fever virus, Boma disease virus, border disease of sheepvirus, borna virus, bovine alphaherpesvirus 1, bovine alphaherpesvirus2, bovine coronavirus, bovine ephemeral fever virus, bovineimmunodeficiency virus, bovine leukemia virus, bovine leukosis virus,bovine mammillitis virus, bovine papillomavirus, bovine papularstomatitis virus, bovine parvovirus, bovine syncytial virus, bovine typeC oncovirus, bovine viral diarrhea virus, Buggy Creek virus, bulletshaped virus group, Bunyamwera virus supergroup, Bunyavirus, Burkitt'slymphoma virus, Bwamba Fever, CA virus, Calicivirus, Californiaencephalitis virus, camelpox virus, canarypox virus, canid herpesvirus,canine coronavirus, canine distemper virus, canine herpesvirus, canineminute virus, canine parvovirus, Cano Delgadito virus, caprine arthritisvirus, caprine encephalitis virus, Caprine Herpes Virus, Capripox virus,Cardiovirus, caviid herpesvirus 1, Cercopithecid herpesvirus 1,cercopithecine herpesvirus 1, Cercopithecine herpesvirus 2, Chandipuravirus, Changuinola virus, channel catfish virus, Charleville virus,chickenpox virus, Chikungunya virus, chimpanzee herpesvirus, chubreovirus, chum salmon virus, Cocal virus, Coho salmon reovirus, coitalexanthema virus, Colorado tick fever virus, Coltivirus, Columbia SKvirus, common cold virus, contagious ecthyma virus, contagious pustulardermatitis virus, Coronavirus, Corriparta virus, coryza virus, cowpoxvirus, coxsackie virus, CPV (cytoplasmic polyhedrosis virus), cricketparalysis virus, Crimean-Congo hemorrhagic fever virus, croup associatedvirus, Cryptovirus, Cypovirus, Cytomegalovirus, cytomegalovirus group,cytoplasmic polyhedrosis virus, deer papillomavirus, deltaretrovirus,dengue virus, Densovirus, Dependovirus, Dhori virus, diploma virus,Drosophila C virus, duck hepatitis B virus, duck hepatitis virus 1, duckhepatitis virus 2, duovirus, Duvenhage virus, Deformed wing virus DWV,eastern equine encephalitis virus, eastern equine encephalomyelitisvirus, EB virus, Ebola virus, Ebola-like virus, echo virus, echovirus,echovirus 10, echovirus 28, echovirus 9, ectromelia virus, EEE virus,EIA virus, EIA virus, encephalitis virus, encephalomyocarditis groupvirus, encephalomyocarditis virus, Enterovirus, enzyme elevating virus,enzyme elevating virus (LDH), epidemic hemorrhagic fever virus,epizootic hemorrhagic disease virus, Epstein-Barr virus, equidalphaherpesvirus 1, equid alphaherpesvirus 4, equid herpesvirus 2,equine abortion virus, equine arteritis virus, equine encephalosisvirus, equine infectious anemia virus, equine morbillivirus, equinerhinopneumonitis virus, equine rhinovirus, Eubenangu virus, European elkpapillomavirus, European swine fever virus, Everglades virus, Eyachvirus, felid herpesvirus 1, feline calicivirus, feline fibrosarcomavirus, feline herpesvirus, feline immunodeficiency virus, felineinfectious peritonitis virus, feline leukemia/sarcoma virus, felineleukemia virus, feline panleukopenia virus, feline parvovirus, felinesarcoma virus, feline syncytial virus, Filovirus, Flanders virus,Flavivirus, foot and mouth disease virus, Fort Morgan virus, FourCorners hantavirus, fowl adenovirus 1, fowlpox virus, Friend virus,Gammaretrovirus, GB hepatitis virus, GB virus, German measles virus,Getah virus, gibbon ape leukemia virus, glandular fever virus, goatpoxvirus, golden shinner virus, Gonometa virus, goose parvovirus,granulosis virus, Gross' virus, ground squirrel hepatitis B virus, groupA arbovirus, Guanarito virus, guinea pig cytomegalovirus, guinea pigtype C virus, Hantaan virus, Hantavirus, hard clam reovirus, harefibroma virus, HCMV (human cytomegalovirus), hemadsorption virus 2,hemagglutinating virus of Japan, hemorrhagic fever virus, hendra virus,Henipaviruses, Hepadnavirus, hepatitis A virus, hepatitis B virus group,hepatitis C virus, hepatitis D virus, hepatitis delta virus, hepatitis Evirus, hepatitis F virus, hepatitis G virus, hepatitis nonA nonB virus,hepatitis virus, hepatitis virus (nonhuman), hepatoencephalomyelitisreovirus 3, Hepatovirus, heron hepatitis B virus, herpes B virus, herpessimplex virus, herpes simplex virus 1, herpes simplex virus 2,herpesvirus, herpesvirus 7, Herpesvirus ateles, Herpesvirus hominis,Herpesvirus infection, Herpesvirus saimiri, Herpesvirus suis,Herpesvirus varicellae, Highlands J virus, Hirame rhabdovirus, hogcholera virus, human adenovirus 2, human alphaherpesvirus 1, humanalphaherpesvirus 2, human alphaherpesvirus 3, human B lymphotropicvirus, human betaherpesvirus 5, human coronavirus, human cytomegalovirusgroup, human foamy virus, human gammaherpesvirus 4, humangammaherpesvirus 6, human hepatitis A virus, human herpesvirus 1 group,human herpesvirus 2 group, human herpesvirus 3 group, human herpesvirus4 group, human herpesvirus 6, human herpesvirus 8, humanimmunodeficiency virus, human immunodeficiency virus 1, humanimmunodeficiency virus 2, human papillomavirus, human T cell leukemiavirus, human T cell leukemia virus I, human T cell leukemia virus II,human T cell leukemia virus III, human T cell lymphoma virus I, human Tcell lymphoma virus II, human T cell lymphotropic virus type 1, human Tcell lymphotropic virus type 2, human T lymphotropic virus I, human Tlymphotropic virus II, human T lymphotropic virus III, Ichnovirus,infantile gastroenteritis virus, infectious bovine rhinotracheitisvirus, infectious haematopoietic necrosis virus, infectious pancreaticnecrosis virus, influenza virus A, influenza virus B, influenza virus C,influenza virus D, influenza virus pr8, insect iridescent virus, insectvirus, iridovirus, Japanese B virus, Japanese encephalitis virus, JCvirus, Junin virus, Kaposi's sarcoma-associated herpesvirus, Kemerovovirus, Kilham's rat virus, Klamath virus, Kolongo virus, Koreanhemorrhagic fever virus, kumba virus, Kysanur forest disease virus,Kyzylagach virus, La Crosse virus, lactic dehydrogenase elevating virus,lactic dehydrogenase virus, Lagos bat virus, Langur virus, lapineparvovirus, Lassa fever virus, Lassa virus, latent rat virus, LCM virus,Leaky virus, Lentivirus, Leporipoxvirus, leukemia virus, leukovirus,lumpy skin disease virus, lymphadenopathy associated virus,Lymphocryptovirus, lymphocytic choriomeningitis virus,lymphoproliferative virus group, Machupo virus, mad itch virus,mammalian type B oncovirus group, mammalian type B retroviruses,mammalian type C retrovirus group, mammalian type D retroviruses,mammary tumor virus, Mapuera virus, Marburg virus, Marburg-like virus,Mason Pfizer monkey virus, Mastadenovirus, Mayaro virus, ME virus,measles virus, Menangle virus, Mengo virus, Mengovirus, Middelburgvirus, milkers nodule virus, mink enteritis virus, minute virus of mice,MLV related virus, MM virus, Mokola virus, Molluscipoxvirus, Molluscumcontagiosum virus, monkey B virus, monkeypox virus, Mononegavirales,Morbillivirus, Mount Elgon bat virus, mouse cytomegalovirus, mouseencephalomyelitis virus, mouse hepatitis virus, mouse K virus, mouseleukemia virus, mouse mammary tumor virus, mouse minute virus, mousepneumonia virus, mouse poliomyelitis virus, mouse polyomavirus, mousesarcoma virus, mousepox virus, Mozambique virus, Mucambo virus, mucosaldisease virus, mumps virus, murid betaherpesvirus 1, muridcytomegalovirus 2, murine cytomegalovirus group, murineencephalomyelitis virus, murine hepatitis virus, murine leukemia virus,murine nodule inducing virus, murine polyomavirus, murine sarcoma virus,Muromegalovirus, Murray Valley encephalitis virus, myxoma virus,Myxovirus, Myxovirus multiforme, Myxovirus parotitidis, Nairobi sheepdisease virus, Nairovirus, Nanirnavirus, Nariva virus, Ndumo virus,Neethling virus, Nelson Bay virus, neurotropic virus, New WorldArenavirus, newborn pneumonitis virus, Newcastle disease virus, Nipahvirus, noncytopathogenic virus, Norwalk virus, nuclear polyhedrosisvirus (NPV), nipple neck virus, O'nyong'nyong virus, Ockelbo virus,oncogenic virus, oncogenic viruslike particle, oncornavirus, Orbivirus,Orf virus, Oropouche virus, Orthohepadnavirus, Orthomyxovirus,Orthopoxvirus, Orthoreovirus, Orungo, ovine papillomavirus, ovinecatarrhal fever virus, owl monkey herpesvirus, Palyam virus,Papillomavirus, Papillomavirus sylvilagi, Papovavirus, parainfluenzavirus, parainfluenza virus type 1, parainfluenza virus type 2,parainfluenza virus type 3, parainfluenza virus type 4, Paramyxovirus,Parapoxvirus, paravaccinia virus, Parvovirus, Parvovirus B 19,parvovirus group, Pestivirus, Phlebovirus, phocine distemper virus,Picodnavirus, Picornavirus, pig cytomegalovirus-pigeonpox virus, Piryvirus, Pixuna virus, pneumonia virus of mice, Pneumovirus, poliomyelitisvirus, poliovirus, Polydnavirus, polyhedral virus, polyoma virus,Polyomavirus, Polyomavirus bovis, Polyomavirus cercopitheci,Polyomavirus hominis 2, Polyomavirus maccacae 1, Polyomavirus muris 1,Polyomavirus muris 2, Polyomavirus papionis 1, Polyomavirus papionis 2,Polyomavirus sylvilagi, Pongine herpesvirus 1, porcine epidemic diarrheavirus, porcine hemagglutinating encephalomyelitis virus, porcineparvovirus, porcine transmissible gastroenteritis virus, porcine type Cvirus, pox virus, poxvirus, poxvirus variolae, Prospect Hill virus,Provirus, pseudocowpox virus, pseudorabies virus, psittacinepox virus,quailpox virus, rabbit fibroma virus, rabbit kidney vaculolating virus,rabbit papillomavirus, rabies virus, raccoon parvovirus, raccoonpoxvirus, Ranikhet virus, rat cytomegalovirus, rat parvovirus, rat virus,Rauscher's virus, recombinant vaccinia virus, recombinant virus,reovirus, reovirus 1, reovirus 2, reovirus 3, reptilian type C virus,respiratory infection virus, respiratory syncytial virus, respiratoryvirus, reticuloendotheliosis virus, Rhabdovirus, Rhabdovirus carpia,Rhadinovirus, Rhinovirus, Rhizidiovirus, Rift Valley fever virus,Riley's virus, rinderpest virus, RNA tumor virus, Ross River virus,Rotavirus, rougeole virus, Rous sarcoma virus, rubella virus, rubeolavirus, Rubivirus, Russian autumn encephalitis virus, SA 11 simian virus,SA2 virus, Sabia virus, Sagiyama virus, Saimirine herpesvirus 1,salivary gland virus, sandfly fever virus group, Sandjimba virus, SARSvirus, SDAV (sialodacryoadenitis virus), sealpox virus, Semliki ForestVirus, Seoul virus, sheeppox virus, Shope fibroma virus, Shope papillomavirus, simian foamy virus, simian hepatitis A virus, simian humanimmunodeficiency virus, simian immunodeficiency virus, simianparainfluenza virus, simian T cell lymphotrophic virus, simian virus,simian virus 40, Simplexvirus, Sin Nombre virus, Sindbis virus, smallpoxvirus, South American hemorrhagic fever viruses, sparrowpox virus,Spumavirus, squirrel fibroma virus, squirrel monkey retrovirus, SSV 1virus group, STLV (simian T lymphotropic virus) type I, STLV (simian Tlymphotropic virus) type II, STLV (simian T lymphotropic virus) typeIII, stomatitis papulosa virus, submaxillary virus, suidalphaherpesvirus 1, suid herpesvirus 2, Suipoxvirus, swamp fever virus,swinepox virus, Swiss mouse leukemia virus, TAC virus, Tacaribe complexvirus, Tacaribe virus, Tanapox virus, Taterapox virus, Tench reovirus,Theiler's encephalomyelitis virus, Theiler's virus, Thogoto virus,Thottapalayam virus, Tick borne encephalitis virus, Tioman virus,Togavirus, Torovirus, tumor virus, Tupaia virus, turkey rhinotracheitisvirus, turkeypox virus, type C retroviruses, type D oncovirus, type Dretrovirus group, ulcerative disease rhabdovirus, Una virus, Uukuniemivirus group, vaccinia virus, vacuolating virus, varicella zoster virus,Varicellovirus, Varicola virus, variola major virus, variola virus,Vasin Gishu disease virus, VEE virus, Venezuelan equine encephalitisvirus, Venezuelan equine encephalomyelitis virus, Venezuelan hemorrhagicfever virus, vesicular stomatitis virus, Vesiculovirus, Vilyuisk virus,viper retrovirus, viral haemorrhagic septicemia virus, Visna Maedivirus, Visna virus, volepox virus, VSV (vesicular stomatitis virus),Wallal virus, Warrego virus, wart virus, WEE virus, West Nile virus,western equine encephalitis virus, western equine encephalomyelitisvirus, Whataroa virus, Winter Vomiting Virus, woodchuck hepatitis Bvirus, woolly monkey sarcoma virus, wound tumor virus, WRSV virus, Yabamonkey tumor virus, Yaba virus, Yatapoxvirus, yellow fever virus, andthe Yug Bogdanovac virus.

Types of virus infections and related disorders that can be treatedinclude, for example, infections due to the herpes family of virusessuch as EBV, CMV, HSV I, HSV II, VZV and Kaposi's-associated humanherpes virus (type 8), human T cell or B cell leukemia and lymphomaviruses, adenovirus infections, hepatitis virus infections, pox virusinfections, papilloma virus infections, polyoma virus infections,infections due to retroviruses such as the HTLV and HIV viruses, andinfections that lead to cellular disorders resulting from and/orassociated with viral infection such as, for example, Burkitt'slymphoma, EBV-induced malignancies, T and B cell lymhoproliferativedisorders and leukemias, and other viral-induced malignancies. Otherneoplasias that can be treated include virus-induced tumors,malignancies, cancers, or diseases that result in a relativelyautonomous growth of cells. Neoplastic disorders include leukemias,lymphomas, sarcomas, carcinomas such as a squamous cell carcinoma, aneural cell tumor, seminomas, melanomas, germ cell tumors,undifferentiated tumors, neuroblastomas (which are also consideredcarcinomas by some), mixed cell tumors, or other malignancies.Neoplastic disorders prophylactically or therapeutically treatable withcompositions of the invention include small cell lung cancers and otherlung cancers, rhabdomyosarcomas, chorio carcinomas, glioblastomamultiformas (brain tumors), bowel and gastric carcinomas, leukemias,ovarian cancers, prostate cancers, osteosarcomas, or cancers that havemetastasized. Diseases of the immune system that are treatable includeHodgkins' disease, the non-Hodgkin's lymphomas including the follicularand nodular lymphomas, adult T and B cell and NK lymphoproliferativedisorders such as leukemias and lymphomas (benign and malignant),hairy-cell leukemia, hairy leukoplakia, acute myelogenous, lymphoblasticor other leukemias, chronic myelogenous leukemia, and myelodysplasticsyndromes. Additional diseases that can be treated or prevented includebreast cell carcinomas, melanomas and hematologic melanomas, ovariancancers, pancreatic cancers, liver cancers, stomach cancers, coloncancers, bone cancers, squamous cell carcinomas, neurofibromas,testicular cell carcinomas, kidney and bladder cancers, cancer andbenign tumors of the nervous system, and adenocarcinomas.

An embodiment of the invention is directed to methods for the treatmentof a patient with a viral infection or a virus-associated neoplasticdisorder comprising the administration of an inducing agent and ananti-viral agent according to a method of the invention. Treatableinfectious diseases include viral infections caused by, withoutlimitation, a hepatitis virus, a retrovirus such as HIV or HTLV, aninfluenza virus, a papilloma virus, a herpes virus (HSV I, HSV II, EBV),a polyoma virus, paramyxovirus, or corona virus, or a “slow viraldisease” such as disease caused by lentiviruses, measles virus (subacutesclerosing panencephalitis), and transmissible spongiformencephalopathies.

Viruses may exist in infected cells as autonomous particles or beintegrated, as, for example, in latent infections. Latent infections maybe periodic, such as HSV-I and II or be continuously productive of virusor virus products, though at fairly low levels. Infections may also belytic, with infectious particles secreted or otherwise extruded orexpelled (virus burst) from cells. Infections may also be of parts of avirus such as, for example, by viral genes or by defective-interferingparticles that are incapable of productive replication on their own, butmay be capable of causing disease.

Cells that can be treated include any cell that becomes infected with avirus or a part of a virus and, preferably, infected by integration.Such cells include cells of the hematopoietic system, such aslymphocytes, erythrocytes and mylocytes, neural cells, andneural-supporting cells, cells of the digestive system, cells of theepithelial system. Cells that contain integrated viral genomes or onlyparts of viral genomes may also be effectively treated.

Anti-neoplastic activity includes cytotoxic (tumor cell death) activity,but also includes, for example, the ability to induce thedifferentiation of transformed cells including cells that compriseneoplasm due to infection (e.g. viral infections such as a humanpapilloma virus, herpes viruses including Herpes Simplex virus type I orII or Epstein-Barr virus, a hepatitis virus, a human T cell leukemiavirus (HTLV) or another retrovirus), and other malignancies. Upondifferentiation, these cells lose their aggressive nature, no longermetastasize, are no longer proliferating, and eventually die and/or areremoved by the T cells, natural killer cells, and macrophages of thepatient's immune system.

The process of cellular differentiation is stimulated or turned on by,for example, the stimulation and/or inhibition of gene specifictranscription. Certain gene products are directly involved in cellulardifferentiation and can transform an actively dividing cell into a cellthat has lost or has a decreased ability to proliferate. An associatedchange of the pattern of cellular gene expression can be observed. Tocontrol this process includes the ability to reverse a malignancy. Geneswhose transcriptional regulation are altered in the presence ofcompositions described herein include the oncogenes myc, ras, myb, jun,fos, abl, and src. The activities of these gene products, as well as theactivities of other oncogenes, are described in Slamon, J. D., et al.1984 Science 224:256-62. Another example of anti-neoplastic activityincludes the ability to regulate the life cycle of the cell, the abilityto repress angiogenesis or tissue regeneration through the blockade orsuppression of factor activity, production or release, the ability toregulate transcription or translation, or the ability to modulatetranscription of genes under angiogenesis, growth factor or hormonalcontrol.

Additional anti-neoplastic activities include the ability to regulatethe cell cycle, for example, by affecting time in and passage through Sphase, M phase, G₁ phase or Gd phase, the ability to increaseintracellular cAMP levels, the ability to inhibit or stimulate histoneacetylation, the ability to methylate nucleic acids, and the ability tomaintain or increase intracellular concentrations of anti-neoplasticagents. The neoplastic disorder may be any disease or malady that couldbe characterized as a neoplasm, a tumor, a malignancy, a cancer, or adisease that results in a relatively autonomous growth of cells.Neoplastic disorders prophylactically or therapeutically treatable viathe methods of the invention include small cell lung cancers and otherlung cancers, rhabdomyosarcomas, choriocarcinomas, glioblastomamultiformas (brain tumors), bowel and gastric carcinomas, leukemias,ovarian cancers, prostate cancers, osteosarcomas, or cancers that havemetastasized. Diseases of the immune system that are treatable carryingout the methods of the invention include Hodgkins' lymphomas and thenon-Hodgkin's lymphomas including the follicular lymphomas, nodularlymphomas, T- and NK-lymphomas, Burkitt's lymphoma, adult T-cellleukemias and lymphomas, hairy-cell leukemia, acute myelogenous,lymphoblastic, or other leukemias, chronic myelogenous leukemia, andmyelodysplastic syndromes. Additional diseases treatable by thecompositions include virally-induced cancers wherein the viral agent isEBV, HPV, HIV, CMV, HTLV-1 or HBV, breast cell carcinomas, melanomas andhematologic melanomas, ovarian cancers, pancreatic cancers, livercancers, stomach cancers, colon cancers, bone cancers, squamous cellcarcinomas, neurofibromas, testicular cell carcinomas, kidney andbladder cancers, tumors of the nervous system, and adenocarcinomas.

C. INDUCING AGENTS

Inducing agents administered according to methods of the inventioninclude, without limitation, short-chain fatty acid (SCFA) derivatives,histone deacetylase (HDAC) inhibitors, phorbol esters, and cytokines.Thus, inducing agents administered according to methods of the inventioninclude SCFA derivatives, for example, without limitation, chemicals ofthe structure R₁—R₂—R₃ or, preferably, R₁—C(O)— R₂—R₃ wherein R₁ isCH_(x), H_(x), NH_(x), OH_(x), SH_(x), COH_(x), CONH_(x), COOH orCOSH_(x); R₂ is CH_(x) or a branched or linear alkyl chain; R₃ isCONH_(x), COSH_(x), COOH, COOR₄, COR₄ or OR₄; R₄ is CH_(x), H_(x),NH_(x), OH_(x), SH_(x) or a branched or linear alkyl chain;phenyl-R₅—R₆—R₇, wherein phenyl is a six carbon benzyl ring or ahydrogenated, hydroxylated or halogenated six carbon ring; R₅ is CH_(x),NH_(x), OH_(x), or SH_(x); R₆ is CH_(x), NH_(x), OH_(x), SH_(x) or abranched or linear alkyl chain; R₇ is CH_(x), H_(x), NH_(x), OH_(x),SH_(x), CONH_(x), COOH, COSH_(x), COOR₈, COR₈ or ORs; R₈ is CH_(x),H_(x), NH_(x), OH_(x), SH_(x) or a branched or linear alkyl chain; andphenyl-R₉—R₁₀ wherein R₉, is CH_(x), NH_(x), OH_(x), SH_(x) or abranched or linear alkyl chain; R₁₀ is CH_(x), H_(x), NH_(x), OH_(x),SH_(x), CONH_(x), COOH, COSH_(x), COOR₁, CUR₁₁ or OR₁₁; and R₁₁ isCH_(x), H_(x), NH_(x), OH_(x), SH_(x) or a branched or linear alkylchain; wherein x is 0, 1, 2 or 3. Preferably, R₄ comprises between 1 to8 carbon atoms and more preferably 1, 2, 3 or 4 carbon atoms.Preferably, R₆ comprises between 1 to 8 carbon atoms and more preferably1, 2, 3 or 4 carbon atoms. Preferably, R₈ comprises between 1 to 8carbon atoms and more preferably 1, 2, 3 or 4 carbon atoms.

Examples of chemical compounds of the structure R₁—R₂—R₃ orR₁—C(O)—R₂—R₃ include acids, amines, monoamides and diamides of butyricacid (H₃C—CH₂—CH₂—COOH), butyric acid ethyl ester (CH₃CH₂CH₂COCH₂CH₃),4,4,4-trifluorobutyric acid (CF₃CH₂CH₂COOH), 2,2-diethyl butyric acid,3,3-dimethyl butyric acid (C₆H₁₂O₂), 3,3-diethyl butyric acid, fumaricacid (HOOCCH═CHCOOH), fumaric acid monomethyl and monoethyl ester,fumaric acid monoamide (C₄H₅O₂N), fumaramide (H₂NCOCHCHCONH₂), succinicacid (HOOCCH₂CH₂COOH) (succinamic acid and succinamide), 2,3-dimethylsuccinic acid and methoxy acetic acid (CH₃CH₂OCH₃).

Examples of chemical compounds of the structure phenyl-R₅—R₆—R₇ includeacids, amines and amides of phenoxyacetic acid (C₆H₅OCH₂COOH;C₆H₅OCH₂COONH₃), 2- and 3-thiophenoxy propionic acid (C₆H₅SCH(CH₃)COOH;C₆H₅SCH₂CH₂COOH), 2- and 3-phenoxy propionic acid (C₆H₅OCH(CH₃)COOH;C₆H₅OCH₂CH₂COOH), 2- and 3-phenyl propionic acid (C₆H₅CH(CH₃)COOH;C₆H₅CH₂CH₂COOH), 4-chlorophenoxy-2-propionic acid (ClC₆OCH₂CH₂CO₂H),methoxy acetic acid (H₃COCH₂CO₂H), and 2-thiophenoxy acetic acid(C₆H₅SCH₂COOH).

Examples of chemical compounds of the structure phenyl-R₉—R₁₀ includeacids, amines and amides of cinnamic acid (C₆H₅CH═CHCOOH), hydrocinnamicacid, dihydro cinnamic acid (C₆H₅CH₂CH₂COOH), a-methyl hydrocinnamicacid or dihydrocinnamic acid, 2,3-dimethyl hydrocinnamic ordihydrocinnamic acid, phenyl acetate ethyl ester(C₆H₅CH(CH₃)CH₂COCH₂CH₃), 2-phenoxypropionic acid (C₆H₅OCH₂CO₂H),phenoxy acetic acid (CH₃CH(OC₆H₅)CO₂H), and 3-phenyl butyric acid(C₆H₅CH(CH₃)CH₂COOH). Additional chemical compounds which may or may notbe included in the above classification scheme include monobutyrin,tributyrin (CH₂(OCOCH₂CH₂CH₃)CH(OCOCH₂CH₂CH₃)CH₂(OCOCH₂CH₂CH₃),ethyl-phenyl acetic acid (CH₃CH₂C₆H₅CH₂COOH), indol-3-propionic acid,indol-3-butyric acid, 1- and 2-methyl cyclopropane carboxylic acid(C₅H₈O₂ and C₆H₈O₂), mercaptoacetic acid (C₂H₄O₂S), N-acetylglycine(C₄H₇O₃N), squaric acid (C₄H₂O₄), 4-trifluorobutanol (C₄H₇OF₃),chloropropionic acid (ClCH₂CH₂CO₂H), 3-trimethyl silyl-1-proposulfonicacid sodium (C₆H₁₅O₃SS), 2-oxopantansane (C₅H₈O₃), isobutylhydroxylamine HCl (C₄H₁₂OCl), 2-methyl butanoic acid (C₅H₁₀O₂),o-benzoyl lactate, n-dimethylbutyric acid glycine amide, o-dimethylbutyric acid lactate, and diethyl butyric acid.

Inducing agents useful in pharmaceutical compositions for the treatmentof virus-associated disorders include, without limitation, propionicacid, butyric acid, succinic acid, fumaric acid monoethyl ester,trifluorobutanol (C₄H₇OF₃), chloropropionic acid (ClCH₂CH₂COOH),isopropionic acid, 2-oxypentasane (CH₃CH₂CH₂C(O)COOH), 2,2- or3,3-dimethyl butyric acid (C₆H₁₂O₂), 2,2- or 3,3-diethyl butyric acid(C₈H₁₆O₂), butyric acid ethyl ester, 2-methyl butanoic acid (C₅H₁₀O₂),fumaric acid (C₄H₄O₃) and amides and salts thereof. Other examplesinclude methoxy acetic acid (H₃C(O)CH₂COOH), methoxy propionic acid,N-acetylglycine (H₃CC(O)NCH₂COOH), mercaptoacetic acid (HSCH₂COOH), 1-or 2-methyl cyclopropane carboxylic acid (C₅H₈O₂), squaric acid(C₄H₂O₄), 2- or 3-phenoxy propionic acid, methoxy butyric acid, phenoxyacetic acid, 4-chloro-2-phenoxy 2-propionic acid, 2- or 3-phenoxybutyric acid, phenyl acetic acid, phenyl propionic acid, 3-phenylbutyric acid, ethyl-phenyl acetic acid, 4-chloro-2-phenoxy-2-propionicacid, n-dimethyl butyric acid glycine amide, o-benzoyl lactic acid,o-dimethyl butyric acid lactate, cinnamic acid, dihydrocinnamic acid(C₆H₅CHCH₃COOH), a-methyl-dihydrocinnamic acid, thiophenoxy acetic acid,and amines, amides and salts of these chemicals.

Inducing agents include retinoic acid, retinol, cytosine arabinoside,phorbols such as the phorbol diester 12-0-tetradecanoylphorbol13-acetate (TPA), teleocidine B, indole alkaloids, cytotoxin, plantlectins from Streptomyces, glucocorticoids such as estrogen andprogesterone, phytohemagglutinin (PHA), bryostatin, growth factors (e.g.PDGF, VEGF, EGF, FGF, NGF, TGF, BCGF), anti-sense nucleic acids (e.g.DNA, RNA or PNA), aptamers (nucleic acid oligonucleotides with secondaryor tertiary structures which bind with high affinity and selectivity toa target molecule), erythropoietin (EPO), the interleukins (IL-1, IL-2,IL-3, etc.), cAMP and cAMP analogs such as dibutyrl cAMP, activin,inhibin, steel factor, interferon, the bone morphogenic proteins (BMBs),hydroxyurea and dimethyl sulfoxide (DMSO). Other inducing agents includeinterferons (e.g. α-, β-, γ-interferon), cytokines such as tumornecrosis factor (TNF), cell receptors, and growth factor antagonists,which may be purified or recombinantly produced.

Inducing agents administered according to the methods of the inventioninclude histone deacetylase (HDAC) inhibitors (including those of thehydroxamic acid class and the benzamide class), DNA methyltransferaseinhibitors, and proteasome inhibitors. HDAC inhibitors, a class ofcompounds that interfere with the function of histone deacetylase,include, without limitation, short-chain fatty acids (butyrate,phenylbutyrate, valproate, AN-9, etc., as described above), hydroxamicacids (for example m-carboxycinnamic acid, bishydroxamic acid, subericbishydroxamic acid, Trichostatin A(7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide),SAHA(suberoyl anilide hydroxamic acid)/Vorinostat, oxamflatin, ABHA,SB-55629, pyroxamide, propenamides, aroyl pyrrolyl hydroxamides,Belinostat/PXD101, Papobinostat, LAQ824(((E)-N-hydroxy-3-[4-[[2-hydroxyethyl-[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]prop-2-enamide),LBH589, TSA), Pivanex, spiruchostatins, cyclic tetrapeptides (forexample, trapoxin A(cyclo((S)-phenylalanyl-(S)-phenylalanyl-(R)-pipecolinyl-(2S,9S)-2-amino-8-oxo-9,10-epoxydecanoyl),),trapoxin B(cyclo((S)-phenylalanyl-phenylalanyl-(R)-prolyl-2-amino-8-oxo-9,10-epoxydecanoyl)),HC-toxin, chlamydocin, diheteropeptin, WF-3161, Cyl-1, Cyl-2, azumamideA), cyclic peptides (for example, FK-228, FR901228), depsipeptides (forexample, romidepsin, FK228((E)-(1S,4S,10S,21R)-7[(Z)-ethylideno]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8,7,6]-tricos-16-ene-3,6,9,22-pentanone),FK228 analogs and derivatives, largazole, largazole analogs andderivatives), peptide antibiotics (apicidin), benzamides (MS275(3-pyridinylmethyl[[4-[[(2-aminophenyl)amino]carbonyl]phenyl]methyl]carbamate,N-(2-Aminophenyl)-4-[N-(pyridine-3ylmethoxycarbonyl)aminomethyl]benzamide),CI994 (4-(Acetylamino)-N-(2-aminophenyl)benzamide), MGCD0103),electrophilic ketones (TPX, AOR, Depudecin), aliphatic acid compounds(for example, butyrate, phenylbutyrate, valproic acid), FR901375,nicotinamide, NAD derivatives, Sirtinol, splitomycin, dihydrocoumarin,naphthopyranone, 2-hydroxynaphthaldehydes, PCYC-0402, PCYC-0403,PCI-24781(3-(dimethylaminomethyl)-N-[2-[4-(hydroxycarbamoyl)phenoxy]ethyl]-1-benzofuran-2-carboxamide),depudecin, tubacin, organosulfur compounds, and dimethyl sulfoxide(DMSO). Other compounds whch may also be administered as inducingagents, which include CHAPs, Scriptaid, Tubacin, JNJ16241199, A-161906,6-(3-Chlorophenylureido)caproic hydroxamic acid, SB939, ITF2357({6-[(diethylamino)methyl]-2-naphthyl}methyl{4-[(hydroxyamino)carbonyl]phenyl}carbamate), 4SC-201, AR-42, OPB-801,RG2833, CUDC-101, JNJ-26481585 (C21H26N6O2), MK0683 (suberoylanilidehydroxamic acid), M344(4-(Diethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide), BML-201(N-(2-aminophenyl)-N′-phenyl-octanediamide), Droxinostat, andpivaloyloxymethyl butyrate.

Useful amines and amides include isobutylhydroxylamine: HCl(C₄H₁₂OCl),fumaric acid monoamide (C₄H₅O₂N), fumaramide (H₂NCOCHCHCONH₂),succinamide and isobutyramide (C₄H₉ON). Salts can be sodium, potassium,calcium, ammonium, lithium or choline such as sodium 3-trimethylsilyl-1-proposulfonic acid (C₆H₁₅O₃SiS:Na). Reagents which may beelectrostatically or covalently bonded with the inducing agent includeamino acids such as arginine (arginine butyrate), glycine, alanine,asparagine, glutamine, histidine or lysine, nucleic acids includingnucleosides or nucleotides, or substituents such as carbohydrates,saccharides, lipids, fatty acids, proteins or protein fragments.Combinations of these salts with the inducing agent can also produceuseful new compounds from the interaction of the combination.

In one embodiment of a method according to the invention, the inducingagent is butyric acid in the form of arginine butyrate, and theantiviral agent is a nucleoside analog. Butyric acid is one of manynaturally-occurring short-chain fatty acids that are generated in thesmall and large bowel by metabolism of carbohydrates. Butyrate is afour-carbon fatty acid with weakly acidic properties and is rapidlyabsorbed and metabolized. Butyrates have shown significant anti-tumoreffects. Sodium butyrate (NAB) has been used clinically in patients withacute myelogenous leukemias and there has now been extensive experiencewith arginine butyrate, a salt of butyrate, in clinical studies for thetreatment of J3-hemoglobinopathies, and more recently with refractorysolid neoplasms (Foss, F. M., et al. 1994 Proc. ASCO 13:162; Sanders, D.A., et al. 1995 Proc. ASCO).

Butyrate and derivatives ofbutyrate including arginine butyrate havedemonstrated several effects upon transformed cell lines in vitro thatinclude decreased DNA replication leading to arrest of cell division inthe G₁ phase, modification of cellular morphology, and alteration ofgene expression consistent with differentiation of a given cell typeexamined (Klehr, D., et al. 1992 Biochemistry 31:3222-29). For example,human tumor cell lines as diverse as colon, breast, melanoma, hepatoma,squamous cell carcinoma of the cervix, endometrial, adenocarcinoma,teratocarcinoma cell lines, leukemic cells (HL-60), and normal humankeratinocytes can all be induced to differentiate in the presence ofbutyrate concentrations ranging from 2-5 mM (Perrin, S. P., et al. 1987Biochem. Biophys. Res. Commun. 148:694-700). The mechanism(s) of actionproposed for these effects upon differentiation are varied and are notfully understood.

Chemical compounds are preferably optically pure with a specificconformation (plus {+} or minus {−}), absolute configuration (R or S),or relative configuration (D or L). Particular salts such as sodium,potassium, magnesium, calcium, choline, amino acid, ammonium or lithium,or combinations of salts may also be preferred, however, certain saltsmay be more advantageous than others. For example, chemical compoundsthat require high doses may introduce too much of a single salt to thepatient. Sodium is generally an undesirable salt, because at high doses,sodium can increase fluid retention resulting in tissue destruction. Insuch instances, lower doses or combinations of different or alternativesalts can be used. For example, compounds of the invention may besubstituted with one or more halogens such as chlorine (Cl), fluorine(F), iodine (I), bromine (Br) or combinations of these halogens. Asknown to those of ordinary skill in the art, halogenation can increasethe polarity, hydrophilicity, or lipophilicity of a chemical compound,which can be a desirable feature, for example, to transform a chemicalcompound into a composition that is more easily tolerated by the patientor more readily absorbed by the epithelial lining of thegastrointestinal tract. Such compositions could be orally administeredto patients.

Therapeutically effective chemical compounds may be created by modifyingany of the above chemical compounds so that after introduction into thepatient, these compounds metabolize into active forms, such as the formsabove, which have the desired effect on the patient. Compounds may alsobe created that are metabolized in a timed-release fashion allowing fora minimal number of introductions that are efficacious for longerperiods of time. Combinations of chemical compounds can also produceuseful new compounds from the interaction of the combination. Suchcompounds may also produce a synergistic effect when used in combinationwith other known or other compounds.

D. ANTIVIRAL AGENTS

Anti-viral agents that can be used in the compositions and methods ofthe provided invention can include, for example, substrates andsubstrate analogs, inhibitors and other agents that severely impair,debilitate or otherwise destroy virus-infected cells. Substrate analogsinclude amino acid and nucleoside analogs. Substrates can be conjugatedwith toxins or other viricidal substances. Inhibitors include integraseinhibitors, protease inhibitors, polymerase inhibitors and transcriptaseinhibitors such as reverse transcriptase inhibitors.

Antiviral agents that can be used in the compositions and methods of theprovided invention can include, for example, ganciclovir,valganciclovir, oseltamivir (Tamiflu), zanamivir (Relenza), abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen,arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir,darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz,emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen,fosamprenavir, foscarnet, fosfonet, fusion inhibitors (e.g.,enfuvirtide), ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, integrase inhibitor, interferon type III, interferon type II,interferon type I, interferon, lamivudine, lopinavir, loviride,maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleosideanalogues, peginterferon alfa-2a, penciclovir, peramivir, pleconaril,podophyllotoxin, protease inhibitor, raltegravir, reverse transcriptaseinhibitor, ribavirin, rimantadine, ritonavir, pyrimidine antiviral,saquinavir, stavudine, synergistic enhancer (antiretroviral), tenofovir,tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine,truvada, valaciclovir (Valtrex), vicriviroc, vidarabine, viramidine,zalcitabine, and zidovudine.

Examples of nucleoside analogs include acyclovir (ACV), ganciclovir(GCV), famciclovir, foscarnet, ribavirin, zalcitabine (ddC), zidovudine(AZT), stavudine (D4T), larnivudine (3TC), didanosine (ddl), cytarabine,dideoxyadenosine, edoxudine, floxuridine, idozuridine, inosine pranobex,2′-deoxy-5-(methylamino)uridine, trifluridine and vidarabine. Examplesof a few protease inhibitors that show particular promise in humantherapy include saquinivir, ritonavir and indinavir. Other anti-viralagents include interferons (e.g. α-, β-, γ-interferon), cytokines suchas tumor necrosis factor (TNF), cell receptors and growth factorantagonists, which can be purified or recombinantly produced.

E. INDUCED GENES INCLUDING VIRAL-ASSOCIATED GENES

Inducing agents (agents that induce expression) may act directly on theviral genome or indirectly through a cellular factor required for viralexpression. For example, viral gene expression can be regulated throughthe regulation of the expression of viral transcription factors such asZTA, RTA, tat, and tax, cellular transcription factors such as AP-1,AP-2, Sp 1, NF-κB, and other transcriptional activators and/orrepressors (factors), co-activators and co-repressors, histoneacetylators and deacetylators, DNA methylases and demethylases,oncogenes or proto-oncogenes, or protein kinase C. These proteins act toregulate and thereby control expression of specific viral and/or othercellular genetic elements. According to the methods of the invention,control over their expression can lead to control over the infection.Other gene products, both viral and cellular in origin, whose expressioncan be regulated with inducing agents include proteases, polymerases,reverse transcriptases, cell-surface receptors, maj orhistocompatibility antigens, growth factors, and combination of theseproducts.

Additional genes whose expression or transcriptional regulation arealtered in the presence of butyric acid include the oncogenes myc, ras,myb, abl and src. The activities of these gene products, as well as theactivities of other oncogenes, are described in Slamon, J. D., et al.1984 Science 224:256-62. Anti-proliferative activity also includes theability to repress tumor angiogenesis through the blockade ofangiogenesis factor activity, production or release, transcriptionalregulation, or the ability to modulate transcription of genes underangiogenesis or growth factor or holinonal control. Either would be aneffective therapy, particularly against both prostatic neoplasia andbreast carcinomas. Further activities that effect transcription and/orcellular differentiation include increased intracellular cAMP levels,inhibition of histone acetylation, and inhibition of genomicmethylation. Each of these activities is directly related to geneexpression, and increased expression can sensitize infected cells to aspecific anti-viral agent.

In some embodiments, inducing agents include arginine butyrate and/orother histone deacetylase inhibitors. Arginine butyrate induces EBV-TKactivity in EBV− immortalized B-cells and patient-derived tumor cells.As latently-infected B-cells do not express TK, exposure of these cellsto agents like arginine butyrate results in a modest induction of lyticreplication and TK expression. TK expression can be used as a point forattack by anti-viral agents, allowing for treatment of latentinfections.

Preliminary in vitro studies according to the invention demonstrate thatinduction of EBV-TK activity in EBV-immortalized B-cells andpatient-derived tumor cells using these drugs is possible, and thatthese previously resistant cells are rendered susceptible to ganciclovirtherapy. Treatment of patients with viral-associated tumors such as EBVwith inducing agents such as arginine butyrate, to induce the expressionof EBV-TK, and GCV, to eliminate EBV-TK expressing tumor cells, is aneffective, non-toxic therapy. This therapeutic regimen does not dependon the associated viral genome being the cause of the tumor. Withoutwishing to be bound by theory, it is believed that just the presence ofthe EBV genome in latent form would make the tumor susceptible to thiscombination protocol.

Butyrate-associated induction of genes has been characterized forvarious cell types, and the genes are consistently in the class ofdifferentiation markers of a cell. For example, in colon cancer celllines, morphologic changes observed in the presence of butyratecorrelate with increased expression of alkaline phosphatase, plasminogenactivator, and CEA, all markers of differentiation. Hepatoma cell linesincrease expression of alpha fetoprotein. Breast cancer cell linesexpress milk-related glycoproteins, epithelial membrane antigens, andincreased lipid deposition. Sodium butyrate can also induce expressionof cellular proteins associated with converting basal keratinacytes intocommitted epithelial cells.

Alteration of expression of certain transcription factors may affectregulation of gene expression and regulation of the cell cycle. In thebreast cancer cell line MCF-7, butyrate induces a block in cellularproliferation that is associated with decreased expression of estrogenand prolactin hormone receptor mRNA expression, thus blocking thepotential growth stimulation by estrogen and prolactin. These effectsare associated with increased expression of the EGF receptor. Butyratealso has been shown to induce down-regulation of c-myc and p53 mRNA andto up-regulate expression of the c-fos transcription factor. In mousefibroblasts, butyrate will block the cell cycle in the G₁ phase. Whenthese cells are stimulated to proliferate with serum, TPA, or insulin,the immediate-early response transcription factors c-myc and c-jun areunregulated. However, the late G₁ phase downstream gene marker cdc-2mRNA is not expressed, and cells are prevented from entering S phase.

The particular combination of inducing agent with anti-viral agent thatis most effective against a specific disorder can be determined by oneof ordinary skill in the art from empirical testing and, preferably,from a knowledge of each agent's mechanism of action. Three suchexamples are as follows. First, many of the RNA viruses such as HIV andother retroviruses require a reverse transcriptase to transcribe theirgenome into DNA. A few of the agents that induce expression or activityof retroviruses and their encoded genes, such as, for example, reversetranscriptase, are known to those of ordinary skill in the art.Anti-viral agents such as nucleoside analogs can be administered to thepatient. Those substrate analogs will be specifically recognized by thereverse transcriptase that, when incorporated into the infected-cellgenome, prevent viral replication and may also result in cell death.Second, many viruses require an active protease to assemble viruscapsids to be packaged with viral genome. Protease inhibitors orproteases that alter cleavage patterns so that packaging cannot occurcan be specifically targeted with an anti-viral agent that comprises anamino acid analog or toxic conjugate. Third, arginine butyrate andisobutyramide enhance expression of viral thymidine kinase and otherviral protein kinases in EBV-infected lymphocytes. Ganciclovir orfamcyclovir, in the presence of the viral thymidine kinase or otherviral kinases, destroys the infected cell. Treatment of infected cellswith both agents, according to the invention, will selectively destroyEBV virus-infected cells. In another aspect, of infected cells with bothagents, according to the invention, will selectively disable or disruptthe viral activity within the cells in vivo.

F. FORMULATIONS, ROUTES OF ADMINISTRATION, AND EFFECTIVE DOSES

Administration of the compositions described herein may be by oral,parenteral, sublingual, rectal, or enteral administration, or pulmonaryabsorption or topical application. Compositions can be directly orindirectly administered to the patient. Indirect administration isperformed, for example, by administering the composition to cells exvivo and subsequently introducing the treated cells to the patient. Thecells may be obtained from the patient to be treated or from agenetically related or unrelated patient. Related patients offer someadvantage by lowering the immunogenic response to the cells to beintroduced. For example, using techniques of antigen matching,immunologically compatible donors can be identified and utilized.

Both inducing and anti-viral agents can be purchased commercially andprepared as a mixed composition using techniques well-known to those ofordinary skill in the art.

Direct administration of a composition may be by oral, parenteral,sublingual, rectal such as suppository or enteral administration, or bypulmonary absorption or topical application. Parenteral administrationmay be by intravenous injection, subcutaneous injection, intramuscularinjection, intra-arterial injection, intrathecal injection,intra-peritoneal injection, or direct injection or other administrationto the site of the neoplasm. An infusion pump (to infuse, for example,the inducing agent into the subject's circulatory system during thefirst period of a cycle of therapy) is generally used intravenously,although subcutaneous, arterial, and epidural infusions are occasionallyused. Injectable forms of administration are sometimes preferred formaximal effect. When long-term administration by injection is necessary,medi-ports, in-dwelling catheters, or automatic pumping mechanisms arealso preferred, wherein direct and immediate access is provided to thearteries in and around the heart and other maj or organs and organsystems. The inducing agent and anti-viral agent may, in an embodimentof the invention, be administered through the same intravenous line.

An effective method of administration to a specific site may be bytransdermal transfusion, such as with a transdermal patch, by directcontact to the cells or tissue, if accessible, such as a skin tumor, orby administration to an internal site through an incision or some otherartificial opening into the body. Compositions may also be administeredto the nasal passages as a spray. Diseases localized to the head andbrain area are treatable in this fashion, as arteries of the nasal areaprovide a rapid and efficient access to the upper areas of the head.Sprays also provide immediate access to the pulmonary system and are thepreferable methods for administering compositions to these areas. Accessto the gastrointestinal tract is gained using oral, enema, or injectableforms of administration. For example, administration of the anti-viralagent to the subject after the first period of a cycle of therapy (i.e.,during the remainder of the cycle, during which only anti-viral agentwithout inducing agent is administered) is preferably oral. As a result,the subject can go through the reminder of the cycle of therapy at home.However, the patient may go through up to about ten, preferably lessthan or equal to six, cycles of therapy.

Administration of the anti-viral agent (amount and frequency) iswell-known to or can be readily determined by the person of ordinaryskill in the art.

As indicated above, orally active compositions are preferred for atleast a portion of the cycle of therapy, as oral administration isusually the safest, most convenient, and economical mode of drugdelivery. Oral administration can, however, be disadvantageous, becausecompositions are poorly absorbed through the gastrointestinal lining.Compounds that are poorly absorbed tend to be highly polar.Consequently, compounds that are effective, as described herein, may bemade orally bioavailable by reducing or eliminating their polarity. Thiscan often be accomplished by formulating a composition with acomplimentary reagent that neutralizes its polarity, or by modifying thecompound with a neutralizing chemical group. Oral bioavailability isalso a problem, because drugs are exposed to the extremes of gastric pHand gastric enzymes. These problems can be overcome in a similar mannerby modifying the molecular structure to be able to withstand very low pHconditions and resist the enzymes of the gastric mucosa such as byneutralizing an ionic group, by covalently bonding an ionic interaction,or by stabilizing or removing a disulfide bond or other relativelylabile bond.

Compounds may also be used in combination with other agents to maximizethe effect of the compositions administered in an additive orsynergistic manner. Compositions may also comprise proteinaceous agentssuch as growth factors and/or cytokines, which are viral inducers. Suchproteinaceous agents may also be aminated, glycosylated, acylated,neutralized, phosphorylated, or otherwise derivatized to formcompositions that are more suitable for the method of administration tothe patient or for increased stability during shipping or storage.Cytokines that may be effective in combination with the compositionsdescribed herein include growth factors such as B cell growth factor(BCGF), fibroblast-derived growth factor (FDGF), granulocyte/macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), macrophage colony stimulating factor (M-CSF), epidermalgrowth factor (EGF), vascular endothelial growth factor (VEGF), plateletderived growth factor (PDGF) nerve growth factor (NGF), stem cell factor(SCF), and transforming growth factor (TGF). These growth factors plus acomposition may further stimulate cellular differentiation and/or theexpression of certain MHC antigens or tumor antigens. For example, BCGFplus a composition may be effective in treating certain B cellleukemias. NGF plus a composition may be useful in treating certainneuroblastomas and or nerve cell tumors. In a similar fashion, otheragents such as differentiating agents may be useful in combination witha composition of the invention to prevent or treat a neoplasticdisorder. Other differentiating agents include B cell differentiatingfactor (BCDF), erythropoietin (EPO), steel factor, activin, inhibin, thebone morphogenic proteins (BMPs), retinoic acid or retinoic acidderivatives such as retinol, the prostaglandins, and TPA.

Alternatively, other cytokines and related antigens in combination witha composition may also be useful to treat or prevent certainvirus-associated cellular disorders, virus infections, and other viraldisorders. Potentially useful cytokines include tumor necrosis factor(TNF), the interleukins IL-1, IL-2, 11-3, IL-4, IL-5, IL-6, etc.,recombinant IL receptors, growth factors, colony stimulating factors,erythropoietin (EPO), the interferon (IFN) proteins IFN-α, IFN-β, andIFN-γ; cyclic AMP including dibutyryl cyclic AMP, hemin, DMSO,hydroxyurea, hypoxanthine, glucocorticoid hormones, and cytosinearabinoside. Therapies using combinations of these agents would be safeand effective therapies against malignancies and other forms of cancer.Combinations of therapies may also be effective in inducing regressionor elimination of a tumor or some other form of cellular disorderresulting from and/or associated with viral infection such ascompositions of the invention plus radiation therapy, toxin ordrug-conjugated antibody therapy using monoclonal or polyclonalantibodies directed against the transformed cells, or specificanti-sense therapy. Effects may be additive, logarithmic, orsynergistic, and methods involving combinations of therapies may besimultaneous protocols, intermittent protocols or protocols that areempirically determined.

Compositions are preferably physiologically stable at therapeuticallyeffective concentrations. Physiological stable compounds are compoundsthat do not break down or otherwise become ineffective upon introductionto a patient prior to having a desired effect. Compounds arestructurally resistant to catabolism, and, thus, physiologically stable,or coupled by electrostatic or covalent bonds to specific reagents toincrease physiological stability. Such reagents include amino acids suchas arginine, glycine, alanine, asparagine, glutamine, histidine, orlysine, nucleic acids including nucleosides or nucleotides, orsubstituents such as carbohydrates, saccharides and polysaccharides,lipids, fatty acids, proteins, or protein fragments. Useful couplingpartners include, for example, glycol, such as polyethylene glycol,glucose, glycerol, glycerin, and other related substances.

A combination therapy can include administering an agent that reducesside effects of treatments, for example, anti-cancer treatments. Acombinational therapy can also include administering an agent thatreduces the frequency of administration of other therapies. The agentcan be an agent that decreases growth of tumor after the anti-cancereffects of other therapies have decreased. The additional agent ortherapy can also be another anti-viral or anti-cancer agent or therapy.

Physiological stability can be measured from a number of parameters suchas the half-life of the compound or the half-life of active metabolicproducts derived from the compound. Certain compounds of the inventionhave in vivo half-lives of greater than about fifteen minutes, greaterthan about one hour, greater than about two hours, and greater thanabout four hours, eight hours, twelve hours, or longer. Although acompound is stable using this criteria, physiological stability can alsobe measured by observing the duration of biological effects on thepatient. Clinical symptoms that are important from the patient'sperspective include a reduced frequency or duration, or elimination ofthe need for transfusions or chelation therapy. Preferably, a stablecompound has an in vivo half-life of greater than about 15 minutes, aserum half-life of greater than about 15 minutes, or a biological effectwhich continues for greater than 15 minutes after treatment has beenterminated or the serum level of the compound has decreased by more thanhalf.

Preferably, compositions are also not significantly biotransformed,degraded, or excreted by catabolic processes associated with metabolism.Although there may be some biotransformation, degradation, or excretion,these functions are not significant, if the composition is able to exertits desired effect.

Compositions are also preferably safe at effective dosages. Safecompositions are compositions that are not substantially toxic (e.g.cytotoxic or myelotoxic), or mutagenic at required dosages, do not causeadverse reactions or side effects, and are well-tolerated. Although sideeffects may occur, compositions are substantially safe if the benefitsachieved from their use outweigh disadvantages that may be attributableto side effects. Unwanted side effects include nausea, vomiting, hepaticor renal damage or failure, hypersensitivity, allergic reactions,cardiovascular problems, gastrointestinal disturbances, seizures, andother central nervous system difficulties, fever, bleeding orhemorrhaging, serum abnormalities, and respiratory difficulties.

Compositions useful for treating viral disorders preferably do notsubstantially affect the viability of a cell such as a normal mammaliancell. Normal cell viability, the viability of an untransformed oruninfected cell, can be determined from analyzing the effects of thecomposition on one or more biological processes of the cell.

Useful combination therapies will be understood and appreciated by thoseof skill in the art. Potential advantages of such combination therapiesinclude the ability to use less of each of the individual activeingredients to minimize toxic side effects, synergistic improvements inefficacy, improved ease of administration or use, and/or reduced overallexpense of compound preparation or formulation. For example, theinducing agent and anti-viral agent may be administered to the subjectin combination with one or more cytokines selected from the groupconsisting, for example, of IL-3, GM-CSF, stern cell factor (SCF), andIL-6.

Administration of the inducing agent and the anti-viral agent to asubject according to a method of the invention may be for prophylaxis ortherapeutic treatment of a confirmed or suspected viral disorder.

Administration may be to an adult, an adolescent, a child, a neonate, aninfant or in utero.

Administration of the inducing agent and the anti-viral agent during thefirst period of a cycle of therapy according to a method of theinvention may be in a single or in separate composition(s).Administration of either agent may be continuous or sporadic, asnecessary. For example, the inducing agent may be administered to thesubject via a continuous infusion throughout the first period of thecycle of therapy. Alternatively, the inducing agent may be administeredto the subject per infusion over a single span of a few to several hoursper day every day throughout the first period of the cycle of therapy.Alternatively, the inducing agent may be administered in a singleparenteral bolus, or orally, daily for several days throughout the firstpart of the cycle, or weekly. Anti-viral agents are administered to thesubject per known or readily determined regimens (for example, one totwo intravenous administrations per day throughout the first period ofthe cycle of therapy, followed by one to two oral administrations perday throughout the remainder of the cycle of therapy). Patients with asuspected or diagnosed viral-associated disorder may only require one ora few cycles of therapy until the disorder has been effectivelyovercome.

Methods for the treatment of viral disorders may include augmenting thetreatment methods of the invention with conventional chemotherapy,radiation therapy, antibody therapy, and/or other forms of therapy. Someconventional chemotherapeutic agents that would be useful in combinationtherapy with methods and compositions of the invention include thecyclophosphamides such as alkylating agents, the purine and pyrimidineanalogs such as mercaptopurine, the vinca and vinca-like alkaloids, theetoposides or etoposide-like drugs, the antibiotics such as deoxyrubocinand bleomycin, the corticosteroids, the mutagens such as thenitrosoureas, antimetabolites including methotrexate, the platinum basedcytotoxic drugs, the hormonal antagonists such as anti-insulin andanti-androgen, the anti-estrogens such as tamoxifen, and other agentssuch as doxorubicin, L-asparaginase, DTIC, mAMSA, procarbazine,hexamethylmelamine, and mitoxantrone. These agents could be givensimultaneously or alternately as defined by a protocol designed tomaximize effectiveness, but minimize toxicity to the patient's body.

Virus-infected cells may also be treated in vivo by administering theinducing agent and the anti-viral agent directly to the patient. Forexample, patients exposed to mutagens, carcinogens, radiation, or othercancer-producing agents may be continuously treated with compositions toinhibit the expected development of a neoplastic condition. Patients whohave been genetically screened and determined to be at high risk for thefuture development of a neoplasia may also be administered compositions,possibly beginning at birth and possibly for life. Both prophylactic andtherapeutic uses are readily acceptable, because these compounds aregenerally safe and non-toxic at effective dosages.

Detrimental interference with one or more of these cellular processesbecomes significant when the process becomes abnormal. Examples ofquantitatable and qualifiable biological processes include the processesof cell division, protein synthesis, nucleic acid (DNA or RNA)synthesis, nucleic acid (principally DNA) fragmentation, and apoptosis.Other processes include specific enzyme activities, the activities ofthe cellular transportation systems such as the transportation of aminoacids by system A (neutral), system B (acidic) or system C (basic), andthe expression of a cell surface protein. Each of these parameters iseasily determined as significantly detrimental, for example, in tissueculture experiments, in animal experiments, or in clinical studies usingtechniques known to those of ordinary skill in the art. Abnormal celldivision, for example, can be mitosis which occurs too rapidly, as in amalignancy, or unstably, resulting in programmed cell death orapoptosis, detected by increased DNA degradation. The determination ofabnormal cell viability can be made on comparison with untreated controlcells. Compositions preferably increase normal cell viability. Increasedcell viability can be determined by those of ordinary skill in the artusing, for example, DNA fragmentation analysis. A decreased amount offragmentation indicates that cellular viability is boosted.Determinations of increased or decreased viability can also be concludedfrom an analysis of the results of multiple different assays. Wheremultiple tests provide conflicting results, accurate conclusions canstill be drawn by those of ordinary skill based upon the cell type, thecorrectness or correlation of the tests with actual conditions, and thetype of composition.

Compositions can be prepared in solution as a dispersion, mixture,liquid, spray, capsule, or as a dry solid such as a powder or pill, asappropriate or desired. Solid forms may be processed into tablets orcapsules or mixed or dissolved with a liquid such as water, alcohol,saline or other salt solutions, glycerol, saccharides or polysaccharide,oil, or a relatively inert solid or liquid. Liquids, pills, capsules ortablets administered orally may also include flavoring agents toincrease palatability. Additionally, all compositions may furthercomprise agents to increase shelf-life, such as preservatives,anti-oxidants, and other components necessary and suitable formanufacture and distribution of the composition. Compositions furthercomprise a pharmaceutically acceptable carrier or excipient. Carriersare chemical or multi-chemical compounds that do not significantly alteror affect the active ingredients of the compositions. Examples includewater, alcohols such as glycerol and polyethylene glycol, glycerin,oils, salts such as sodium, potassium, magnesium, and ammonium, fattyacids, saccharides, or polysaccharides. Carriers may be singlesubstances or chemical or physical combinations of these substances.

Compositions administered as part of the methods of the inventioncomprise an inducing agent to induce expression of a gene product in avirus-infected cell and an anti-viral agent whose anti-viral activityrelates to or is directed to the expressed product. The gene productexpressed may be a viral enzyme or a cellular enzyme or activity that islargely expressed in virus-infected cells. Expression products that canbe targeted include enzymes involved with DNA replication, which may beeither for repair or replication of the genome, assembly of completevirus particles, generation of viral membrane or walls, RNAtranscription or protein translation, or combinations of theseactivities. Interference with these processes can be performed byinducing and then acting on an enzyme and, preferably, a critical enzymein the process.

Administration Therapy

Preferably, compositions administered contain chemicals that aresubstantially non-toxic. Substantially non-toxic means that thecomposition, although possibly possessing some degree of toxicity, isnot harmful to the long-term health of the patient. Although the activecomponent of the composition may not be toxic at the required levels,there may also be problems associated with administering the necessaryvolume or amount of the final form of the composition to the patient.For example, if the composition contains a salt, although the activeingredient may be at a concentration that is safe and effective, therecan be a harmful build-up of sodium, potassium, or another ion. With areduced requirement for the composition or at least the active componentof that composition, the likelihood of such problems can be reduced oreven eliminated. Consequently, although patients may suffer minor orshort term detrimental side-effects, the advantages of taking thecomposition outweigh the negative consequences.

Treatment of a patient may be therapeutic and/or prophylactic.Therapeutic treatment involves administration of an inducing agent andan anti-viral agent according to a method of the invention to a patientsuffering from one or more symptoms of or having been diagnosed as beingafflicted with a viral disorder. Relief and even partial relief from oneor more of the symptoms may correspond to an increased life span or,simply, an increased quality of life. Further, treatments that alleviatea pathological symptom can allow for other treatments to beadministered.

Prophylactic treatments involve administration of an inducing agent andan anti viral agent according to a method of the invention to a patienthaving a confirmed or suspected viral disorder without having any overtsymptoms. Administration can begin at birth and continue, if necessary,for life. Both prophylactic and therapeutic uses are readily acceptable,because these compounds are generally safe and non-toxic.

Treatments Aids

Aids for the treatment of human viral disorders and virus-associatedcellular and neoplastic disorders contain compositions described hereinin predetermined amounts, which can be individualized in concentrationor dose for a particular patient. Compositions, which may be liquids orsolids, are placed into reservoirs or temporary storage areas within theaid. At predetermined intervals, a set amount of one or morecompositions is administered to the patient. Compositions to be injectedmay be administered through, for example, infusion pumps, mediports, orin-dwelling catheters. Aids may further comprise mechanical controls orelectrical controls devices, such as a programmable computer or computerchip, to regulate the quantity or frequency of administration topatients. Examples include both single and dual rate infusers andprogrammable infusers. Delivery of the composition may also becontinuous for a set period of time. Aids may be fixed or portable,allowing the patient as much freedom as possible. The use of a portableaid allows the patient to receive at least a portion of the treatmentregimen outside of the hospital.

Administration Schedule

Administration of one or more agents (e.g, a viral inducing agent and/oran antiviral) can be intermittent; for example, administration can beonce every two days, every three days, every five days, once a week,once or twice a month, and the like. The amount, forms, and/or amountsof the different forms can be varied at different times ofadministration.

Pulsed administration of one or more pharmaceutical compositions can beused for the treatment or prevention of a viral-induced inflammatorydisease. Pulsed administration can be more effective than continuoustreatment as pulsed doses can be lower than would be expected fromcontinuous administration of the same composition. Each pulse dose canbe reduced and the total amount of drug administered over the course oftreatment to the patient can be minimized.

With pulse therapy, in vivo levels of an agent can drop below that levelrequired for effective continuous treatment. Pulsed administration canreduce the amount of the composition administered to the patient perdose or per total treatment regimen with an increased effectiveness.Pulsed administration can provide a saving in time, effort and expenseand a lower effective dose can lessen the number and severity ofcomplications that can be experienced by a subject. As such, pulsing canbe more effective than continuous administration of the samecomposition.

Individual pulses can be delivered to a subject continuously over aperiod of several hours, such as about 2, 4, 6, 8, 10, 12, 14 or 16hours, or several days, such as 2, 3, 4, 5, 6, or 7 days, or from about1 hour to about 24 hours or from about 3 hours to about 9 hours.Alternatively, periodic doses can be administered in a single bolus or asmall number of injections of the composition over a short period oftime, for example, less than 1 or 2 hours. For example, argininebutyrate can be administered over a period of 4 days with infusions forabout 8 hours per day or overnight, followed by a period of 7 days of notreatment.

The interval between pulses or the interval of no delivery can begreater than 24 hours or can be greater than 48 hours, and can be foreven longer such as for 3, 4, 5, 6, 7, 8, 9 or 10 days, two, three orfour weeks or even longer. The interval between pulses can be determinedby one of ordinary skill in the art. The interval between pulses can becalculated by administering another dose of the composition when thecomposition or the active component of the composition is no longerdetectable in the patient prior to delivery of the next pulse. Intervalscan also be calculated from the in vivo half-life of the composition.Intervals can be calculated as greater than the in vivo half-life, or 2,3, 4, 5 and even 10 times greater than the composition half-life.Intervals can be 25, 50, 100, 150, 200, 250 300 and even 500 times thehalf life of the chemical composition.

The number of pulses in a single therapeutic regimen can be as little astwo, but can be from about 5 to 10, 10 to 20, 15 to 30 or more. Subjects(e.g., patients) can receive one or more agents (e.g., drugs) for lifeaccording to the methods of this invention. Compositions can beadministered by most any means, and can be delivered to the patient asan injection (e.g. intravenous, subcutaneous, intraarterial), infusionor instillation, and more preferably by oral ingestion. Various methodsand apparatus for pulsing compositions by infusion or other forms ofdelivery to the patient are disclosed in U.S. Pat. Nos. 4,747,825;4,723,958; 4,948,592; 4,965,251 and 5,403,590.

In one embodiment, the inducing agent and the anti-viral agent areadministered for about five days, and the anti-viral agent issubsequently administered without the inducing agent for an additionalperiod of about sixteen days for a total cycle of about 21 days. Cyclesof treatment may occur in immediate succession or with an interval of notreatment between cycles.

A pharmaceutical composition comprising a viral inducing agent can beadministered to a subject before a pharmaceutical composition comprisingan antiviral agent is administered to the subject. A pharmaceuticalcomposition comprising a viral inducing agent can be co-administered toa subject with a pharmaceutical composition comprising an antiviralagent. A pharmaceutical composition comprising a viral inducing agentcan be co-administered with a pharmaceutical composition comprising anantiviral agent and a pharmaceutical composition comprising one or moreaddition agents. The pharmaceutical compositions can be provided bypulsed administration. For example, a pharmaceutical compositioncomprising a viral inducing agent can be administered to a subject,followed by administration of a pharmaceutical composition comprising anantiviral agent to the subject after an interval of time has passed, andthis order of administration the same or similar time interval can berepeated, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moretimes.

INCORPORATION BY REFERENCE

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference and may be employed in the practice ofthe invention. More generally, documents or references are cited in thistext, either in a Reference List before the claims, or in the textitself; and, each of these documents or references (“herein citedreferences”), as well as each document or reference cited in each of theherein cited references (including any manufacturer's specifications,instructions, etc.), is hereby expressly incorporated herein byreference.

EXAMPLES Example 1 Phase 1 Study of AB Plus Ganciclovir in Patients withEBV Associated Lymphoid Malignancies

Fifteen patients with EBV-associated lymphoid malignancies, who hadhistologically confirmed lymphoid neoplasms that were EBV+, were treatedwith AB and GCV. Prior therapies (varying in different subjects)included rituximab, chemotherapy, chemoradiotherapy and bone marrowtransplant. GCV was administered at a rate of 5 mg/kg intravenously (IV)over 1 hour twice per day, and continued throughout the cycle. AB wascontinuously infused at a starting dose of 500 mg/kg/day. Doseescalation was continued as follows until MTD was established:

Level 1: 500 mg/kg/day IV for 2 days

Level 2: 1000 mg/kg/day IV for 2 days

Level 3: 1500 mg/kg/day IV for 2 days

Level 4: 2000 mg/kg/day IV until day 21

A total of 15 patients were evaluated for anti-tumor response (Table 1).A complete response (CR) was defined as disappearance of detectablemalignant disease on imaging or physical examination (e.g., for skinlesions or tonsillar masses). A partial response (PR) was defined as a50% decrease in tumor size (the sum of the product of the largestperpendicular diameters) or measurable lesions chosen for analysis priorto beginning of treatment. For lesions which could only be measured in 1dimension, such as skin (cutaneous T cell lymphoma), a greater than 50%decrease in the largest dimension qualified as a PR. For the 3 patientswho died from co-morbidities, anti-tumor responses were confirmedpathologically at autopsy.

Four patients were classified as achieving CRs, including 2 with PTLD, 1with extranodal NK/T cell lymphoma and 1 with peripheral T-celllymphoma. Three of these patients died after completing therapy as aresult of co-morbid conditions and complications presumed related totumor regression. Autopsy examination of 2 subjects revealed apparentcomplete disappearance of tumor, while the third patient demonstratedsignificant necrosis of residual lymphoma at autopsy.

Six patients were classified as partial responses (PRs), including 3with PTLD, 1 with diffuse large-cell B-cell lymphoma, 1 with extranodalNK/T-cell lymphoma and 1 with subcutaneous panniculitis-like T-celllymphoma.

The remaining 5 patients were classified as non-responders (NR).

TABLE 1 Treatment Courses in Patients With EBV Associated LymphoidMalignancies Treated with AB and GCV Patient HD/HTD¹ Outcome, Number No.Cycles (mg/kg/day) 1 cycle Adverse Events 1 <1, 15 d  500 CR confusion;diarrhea; emesis; rejection of lung transplant* 2 <1, 16 d 1800 CRconfusion; mucositis; headache; nausea; vomiting; abdominal pain 3 <1,19 d 2000 PR confusion; mucositis; headache; nausea/vomiting; tumorregression preceding bowel perforation* 4 1 2000 PR confusion:nausea/vomiting; anorexia 5 1 2000 NR confusion; restlessness;somnolence; nausea; vomiting; abdominal pain; vision change; orthostasis6 1 1000/1000 NR Headache; nausea/vomiting; abdominal pain;thrombocytopenia 7 1 2000/1500 CR Lethargy/stupor/confusion;hypotonia/hypoesthesia; fungal infection/mucositis; tumor lysis leadingto hemorrhage* 8 1 1500/1000 PR Acoustic hallucinations; somnolence;hypokalemia; sepsis; Deep Vein Thrombosis 9 1 2000/2000 CR Confusion;fatigue; elevated BUN; tumor lysis leading to pancreatitis/hepatitis* 101 1000/800  NR Elevated BUN, encephalopathy 11 <1, 8 d 1500/1500 PRDiarrhea; hepatomegaly 12 1 2000/2000 NR Nausea; pneumonia; portinfection 13 3 938/938 PR Nausea; anorexia; weight loss; anemia;thrombocytopenia; lethargy; insomnia; hypokalemia 14 <1, 19 d 1250/1250NR Sinus, throat, back pain; thrombocytopenia; hypokalemia; lethargy 15<1, 5 d 1000/1000 PR Lethargy; increased dyspnea; polymicrobialpneumonia/acute respiratory distress syndrome *fatal AE; ¹HD/HTD-highestdose/highest tolerated dose.

In summary 10 out of 15 patients showed a degree of response totreatment of AB in combination with the antiviral ganciclovir.

Example 2 Phase II Trial of Low-Dose Arginine Butyrate andGanciclovir/Valganciclovir in EBV(+) Lymphoid Malignancies

It has previously been found that continuous infusion of inducing agent,for example, arginine butyrate, may not be necessary to maintain viralthymidine kinase expression and sensitization to anti-viral agents inEBV-associated tumors, but that, in fact, cells that survived initialexposure to the inducing agent plus the anti-viral agent remainedsusceptible to further cycles of combination treatment (Ghosh, S. K., etal. 2007 Blood Cells, Molecules, and Diseases 38:57-65, incorporatedherein in its entirety). However, it was neither anticipated norexpected that after some first period of treatment with inducing agentand anti-viral agent, one could continue the anti-viral treatmenteffectively within a cycle of therapy without continued administrationof the inducing agent (continued administration including continuedperiods of pulsing throughout).

A clinical trial was instituted utilizing a 5-day infusion of argininebutyrate and 21 days of ganciclovir/valganciclovir for EBV+ lymphomasand Post-transplant Lymphoproliferative Disorder (PTLD). The firstpatient enrolled in the protocol (with Rituximab-refractory PTLDfollowing a cord stem cell transplantation for Hodgkin Disease)tolerated the treatment regimen well, with resolution of cough withinthree days and a decrease in LDH levels.

Treatment with arginine butyrate (AB) was administered in ahospital/inpatient basis. The subject was a 32 year old with EBV-relatedpost-transplant lymphoma who had failed multiple therapies(chemotherapy, Rituxan). The subject received AB 1,000 mg/kg/doseintravenously for 5 days (day 1-5). The dose was given continuously over24 hours. AB was given through a long IV line or port due tohypertonicity. Ganciclovir at 5 mg/kg IV over 1 hour was given twice aday for five days (day 1-5). Valganciclovir 900 mg was given orallytwice per day for 16 days (day 6-21). At the end of the 21-day cycle,imaging studies were done to determine response and revealed eliminationof nearly all tumor masses (FIG. 1). Four of six target lesions resolvedcompletely, and two additional lesions decreased in size. (Table 2) Thesubject's symptoms of fever and cough resolved for first time in 9weeks. Measure of the tumor marker serum LDH was reduced from 899 to 328(normal). Additionally, EBV, CMV, and HH6 viral load becameundetectable. These findings indicate that a shorter, morepatient-accessible regimen of the virus-target therapeutic strategy ismore efficacious.

TABLE 2 Quantification of tumor response evaluated by CT Scan. Tumordimensions in cm. Pre-Treatment Post-Treatment Dimension DimensionDimension Dimension Location 1 2 1 2 R. Upper lobe 0.7 0.7 None None R.Mid Lobe 1.1 1.1 None None R. Lower Lobe 1.4 0.8 0.8 0.6 L. Upper Lobe0.9 0.8 None None L. Lower Lobe 0.9 0.6 0.6 0.5 Lingular 0.9 0.7 NoneNone Hepatic Seg. 6 1.1 1.1 None None Hepatic Seg. 8 1.0 0.7 None NoneL. Ant. Abd. Wall 1.1 1.9 None None R. Ant. Abd. Wall 0.9 0.5 0.9 0.5

Example 3 Analysis of Efficacy of the Herpes Anti-Virals

There are 12 mammalian HDACs, and any one of which might be required forrepression of TK gene during latency in tumors. HDAC isozyme-specificsiRNAs were used to to knockdown individual HDACs in tumor linesexpressing latent EBV to determine which one of them inducesreactivation of TK from latency, rendering it susceptible toanti-virals.

The EBV-positive B lymphoma cell line P3HR1 was used throughout theseassays. The P3HR1 cell line was originally derived from Burkitt'slymphoma patient. EBV maintains a latent state of replication in thiscell line. Cells were maintained in RPMI 1640 with 10% fetal bovineserum containing 100 U penicillin per ml and 100 μg streptomycin per ml.The HDAC inhibitors used were from five different classes: a) shortchain fatty acids, b) hydroxamic acids, c) benzamides, d) cyclictetrapeptides, and e) largazoles.

To measure the relative level of TK mRNA in various total RNApreparations, reverse transcription and quantitative PCR using real timePCR technology was used. Five micrograms of total RNA wasreverse-transcribed using random hexamer primers and Superscript IIIcDNA synthesis kit (Invitrogen). The cDNA was diluted to a final volumeof 60 μl with sterile water, 8 μl of which was then used in each realtime PCR reaction in an ABI 7500 Sequencher using SYBR-Green technology.Primers used for the amplification of TK were EBV-TK1-F:5′-AGATGACGACGGCCTCTACCA-3′; EBV-TK1-R: 5′-CCTCCTTCTGTGCACGAAGT-3′. Theβ-actin mRNA level in those samples were determined similarly usingβ-actin-specific primers Actin/hu-F: 5′-GCTCGTCGTCGACAACGGCTC-3′;Actin/hu-R: 5′-CAAACATGATCTGGGTCATCTTCTC-3′. The relative level of TKexpression in a sample was calculated following normalization of β-actinexpression level.

Toxicity assays with two anti-herpesvirus drugs, Gancicovir (GCV) andPenciclovir (PCV), treated to P3HR1 cells alone was conducted as acontrol. A total of 3×10⁵ P3HR1 cells were incubated with variousconcentrations of GCV or PCV and incubated for 6 days. Viable cellcounts were measured and toxicity was expressed as percentage of cellgrowth compared to untreated cells. As shown in FIGS. 2A and 2B, PCV wasless toxic to the cells compared to GCV. The effect of 40 μM GCV and PCVin combination treatment approach with 1.0 mM Na-butyrate in P3HR1 cellswas compared (FIG. 2C). Inhibition of cell growth with 40 μM PCV (76%)was much less than with 40 μM GCV (38%). This lower level of inhibitionof cell growth with PCV did not change significantly when the drug wasused at higher concentrations (FIG. 2D).

Example 4 Analysis of Efficacy of HDAC Inhibitors A. Short Chain FattyAcids

Two SCFA HDAC inhibitors, Na-butyrate (NaB) and valproic acid (VA) weretested. In a combination treatment approach, NaB+GCV reduced growth ofEBV-positive P3HR1 cells significantly (up to 50% more) compared tocells treated with NaB or GCV alone (FIG. 3A). The optimal concentrationof NaB for this purpose was found to be 1.0 mM. Responses from 1.0 mMNaB was used as a control for interpreting results in this experiment.At a higher concentration NaB alone reduces cell growth to a significantdegree, and the synergistic effects of GCV are lost at thoseconcentrations of NaB. The other HDACi used in this experiment, VA, alsohad very similar activity (FIG. 3B). Analysis of TK mRNA level by RT andreal-time PCR however showed that VA was less efficient than NaB ininducing TK expression (FIG. 3C).

B. Hydroxamic Acids

A total of five different HDAC inhibitors from the hydroxamic acid groupwere examined as combination therapies. These inhibitors includescriptaid, SAHA, panobinostat-LHB589, belinostat-PXD 101 and oxamflatin.

Scriptaid: Scriptaid showed strong synergistic effect with GCV inreducing cell growth of P3HR1 cells, especially at 500 nM and 1 μMconcentrations (FIG. 4A).

SAHA-Vorinostat: The combination treatment experiment with SAHA showedthat it was less effective in reducing cell growth when combined with anantiviral agent compared to butyrate (FIG. 5A) although it did induce TKexpression at a higher level than that seen with efficientconcentrations of butyrate (1.0 mM) (FIG. 5B).

LHB589-Panobinostat: The growth inhibitory activity of LHB589 at a 50 nMconcentration was comparable to that of NaB at 1.0 mM (FIG. 6A). Whenthe cells were treated for 3 days or longer, LHB589 was extremely toxicto the cells at any concentrations 100 nM or above. Although whentreated for 24 h only, cells survived well even at a concentration of 5μM. TK expression level in presence of LHB589 was quite high compared tooptimum concentration of NaB (2.5 mM) (FIG. 6B).

PXD101-Belinostat: PXD101 induced high level of TK expression at the 5μM concentration. (FIG. 7).

Oxamflatin: Oxamflatin showed synergistic activity with GCV towardsreducing cell growth. At a 200 nM concentration, the activity level(growth suppression) was more than what typically seen with 1.0 mM NaB.(FIG. 8)

C. Cyclic Tetrapeptide

Apicidin: The cyclic tetrapeptide group of HDACi examined was apicidin.A toxicity assay with apicidin alone showed that concentrations ofapicidin higher than 200 nM was quite toxic to the cells. Thecombination treatment assay (FIG. 9) showed that at 100 nM and 200 nMconcentrations, apicidin reduced cell growth by 40-50% over cellstreated with apicidin alone. However, the 200 nM concentration cellgrowth was significantly retarded without any GCV and a 500 nMconcentration was very toxic to the cells.

D. Benzamide

Experiments show that the benzamide class of HDAC inhibitors wereextremely potent in sensitizing P3HR1 cells to GCV-mediated effects. Asshown below (FIG. 10A) a 500 nM concentration of MS-275 was as efficientas 1.0 mM NaB. Higher concentrations were extremely toxic to the cells.Interestingly, MS-275 also strongly induced TK expression at 500 nM andhigher concentrations (FIG. 10B). TK expression was also induced at only6 hr post treatment. Based on these results, an even shorter exposure toMS-275 was examined to see if it would be sufficient to sensitize P3HR1cells to GCV-mediated killing. Cells with were treated with MS-275+GCVfor shorter time periods of 24 hr or 48 hr (as opposed to 72 hr) andthen further incubated in presence of GCV for up to 6 days, at whichtime the viable cell counts were enumerated. As shown in FIG. 10C, evenat just 24 hr exposure to MS-275 sensitized the cells to GCV-mediatedeffects as efficiently as a 72 hr continuous treatment. This furtherdemonstrates that MS-275 is very effective sensitizing agent forcombination treatment studies.

E. Largazole

Largazole is a member of macrocyclic depsipeptide that was originallyisolated from coral reef cyanobacteria. Largazole is a potent HDACinhibitor with specificity towards HDAC class 1 and 2 only.Additionally, largazole has very low IC 50 and HDAC isozyme specificity.16 different analogs of the largazole were tested (ab6-113b, ab6-113a,ab6-123a, ab6-123b, ab6-164b, ab6-156b, 232a, 233a, 238a, 233b, 234b,235b, 234a, 235a, 237a, 212b, TLN1 357, TNL2 380, ART01) for synergisticcell killing activity in combination with GCV (FIGS. 12A, 12B, 12C, 12Dand 12E). 13 largazole derivatives were tested both in combinationtreatment approach and also for their ability to induce EBV TK (FIG.12F). Several of the largazoles showed potent cell killing activity incombination with GCV.

Example 5 Analysis of Efficacy of Combination Treatment withHIV-Infected Cells

Virus production (p24 release) was examined in an HIV-1-infectedmonocyte line. Cells were treated or not treated with HDAC-inhibitorsand other compounds. P24 release expressed as optical density (“OD”)(FIG. 13), and then converted to pg of protein (FIG. 14). Argininebutyrate, phorbol myristate acetate (PMA), trichostatin A (TSA), LHB589,apicidin (API) and largazole (LARG) are shown to be active, whereas2,2-dimethyl butyrate (ST20) and 2-(quinazolin-4-ylamino)butanoic acid(RB3) increased viral production at levels similar to the control ofvehicle alone. DMSO was vehicle for some of the compounds tested.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A method for treating neoplasma associated with Epstein-Barrinfection in a subject, comprising administering to the subject aninducing agent to induce expression of a viral gene product in avirus-infected cell of the subject and an anti-viral agent whoseanti-viral activity is directed to the viral gene product expressed,wherein said inducing agent is a HDAC inhibitor, wherein the HDACinhibitor is a hydroxamic acid derivative, and wherein said subject isnot treated with radiation therapy.
 2. The method according to claim 1,wherein said neoplasia associated with Epstein-Barr virus infection is acancer associated with Epstein-Barr virus infection.
 3. The methodaccording to claim 1, wherein said neoplasia is selected from the groupconsisting of lymphoma, Hodgkin disease, Burkitts lymphoma,post-transplantation lymphoproliferative disease, viral associatedlymphoproliferative disease, hemophagocytic syndrome, nasopharyngealcarcinoma, gastric carcinoma, and breast cancer.
 4. The method accordingto claim 1, wherein said neoplasia is lymphoma.
 5. The method of claim1, wherein the inducing agent induces expression of a viral gene productin a virus-infected cell of the subject, wherein the viral gene productis a viral enzyme, an oncogene or proto-oncogene, a transcriptionfactor, a protease, a polymerase, a reverse transcriptase, a cellsurface receptor, a structural protein, a major histocompatibilityantigen, a growth factor, or a combination thereof.
 6. The method ofclaim 4, wherein the viral gene product is a viral enzyme selected froma thymidine kinase (TK) or protein kinase (PK).
 7. The method of claim1, wherein said inducing agent is panobinostat (LBH589).
 8. The methodof claim 1 wherein said inducing agent induces viral TK expression. 9.The method of claim 1, wherein said inducing agent is administered at adose of about 0.1 to about 2000 mg/kg/day or about 1 to about 100mg/m²/day.
 10. The method of claim 1, wherein said anti-viral agent isselected from the group consisting of an interferon, an amino acidanalog, a nucleoside analog, an integrase inhibitor, a proteaseinhibitor, a polymerase inhibitor, and a transcriptase inhibitor. 11.The method of claim 1, wherein the anti-viral agent is a nucleosideanalog selected from the group consisting of acyclovir (ACV),ganciclovir (GCV), valganciclovir, famcyclovir, penciclovir (PCV),foscarnet, ribavirin, zalcitabine (ddC), zidovudine (AZT), stavudine(D4T), lamivudine (3TC), didanosine (ddl), cytarabine, dideoxyadenosine,edoxudine, floxuridine, idozuridine, inosine pranobex,2′-deoxy-5-(methylamino)uridine, trifluridine and vidarabine.
 12. Themethod of claim 1, wherein said anti-viral agent is ganciclovir orvalganciclovir.
 13. The method of claim 1, wherein the inducing agent isan HDAC inhibitor having inhibitory activity at 500 nM concentration.14. The method of claim 1, wherein the inducing agent is an HDACinhibitor capable of inducing TK expression at 500 nM.
 15. The method ofclaim 1, wherein the inducing agent is an HDAC inhibitor capable ofinducing TK expression within 6 hours of treatment.
 16. The method ofclaim 1, wherein said inducing agent is administered orally.
 17. Themethod of claim 1, wherein said inducing agent and said anti-viral agentare administered for at least one cycle of therapy, said cyclecomprising: i. administering the inducing agent and the anti-viral agentto the subject over a first period of time; and ii. continuing theadministration of the anti-viral agent to the subject for a secondperiod; wherein said second period represents the remainder of thecycle;
 18. The method of claim 15, wherein during the first period theanti-viral agent and inducing agent are administered in the samecomposition.
 19. The method of claim 15, wherein said first period oftime is less than or equal to one-half of the length of the cycle. 20.The method of claim 17, wherein said first period of time is less thanor equal to about 5 days, and wherein said cycle is less than or equalto about 21 days.